Pseudotyped lentiviral vectors and uses thereof

ABSTRACT

A vector system that will produce a pseudotyped lentiviral vector that can be used to deliver a desired gene is disclosed. The vector constructs that are described include a number of modifications that enhance the safety of the vector. The vector can be used to more specifically target cells for expression of certain genes.

The following application is a continuation of PCT/US00/06971, filed 16Mar. 2000, which is an international filing of provisional application60/124,641, filed 16 Mar. 1999.

The present invention was funded in part by National Institutes ofHealth grants 5PO HL59316-02 and 5P30 AI 28691-10, and the U.S.Government has certain rights thereto.

FIELD OF THE INVENTION

The present invention is directed to a vector system wherein multiplelentiviral vectors are used to transfer nucleic acid segments to hostcells. Preferably the system uses an inducible expression system toexpress the nucleic acid segments, and the lentiviral vectors arepseudotyped lentiviral vectors.

BACKGROUND OF THE INVENTION

In recent years considerable effort has been directed at applying genedelivery techniques. That term describes a wide variety of methods usingrecombinant biotechnology techniques to deliver a variety of differentmaterials to a cell. These methods include, for example, vectors such asviral vectors, liposomes, naked DNA, adjuvant-assisted DNA, gene gun,catheters, etc. The different techniques used depend in part upon thegene being transferred and the purpose therefore. Thus, for example,there are situations where only a short-term expression of the gene isdesired in contrast to situations where a longer term, even permanentexpression of the gene is desired.

Vectors that have been looked at include both DNA viral vectors and RNAviral vectors. For example, DNA vectors include pox vectors such asorthopox or avipox vectors (see, e.g., U.S. Pat. No. 5,656,465), herpesvirus vectors, such as herpes simplex I Virus (HSV) vectors [Geller, A.I. et al., J. Neurochem. 64:487 (1995); Lim, F., et al., DNA Cloning:Mammalian Systems, D. Glover, Ed., Oxford Univ. Press, Oxford, England(1995); Geller, A. I. et al., Proc. Natl. Acad. Sci., U.S.A. 90:7603(1993)]: Adenovirus vectors [Legal Lasalle et al., Sci. 259-988 (1993);Davidson et al., Natl. Genet. 3:219 (1993); Yang et al., J. Virol.,69:2004 (1995)]; and Adeno Associated Virus Vectors [Kaplitt, M. G., etal., Nat. Genet. 8;148 (1994)]. Retroviral vectors include Moloneymurine leukemia viruses (MMLV) and human immunodeficiency viruses (HIV)[See, U.S. Pat. No. 5,665,577].

Recently, a great deal of attention has been focused on problems thatmay have arisen with clinical trials using gene delivery techniques.Many of the criticisms that have been raised do not address the value ofsuch work, but rather failures to follow reporting procedures whereadverse reaction and deaths occur. The individuals now receiving genetherapy are typically ill. Many have proved refractory to othertreatment protocols. A number of these individuals have extremely poorlife expectancies. Thus, as with other trials of patients in such poorhealth, problems can arise. It would be desirable to further improvegene delivery systems to reduce such problems.

Further, while attention has been focused on the use of viral vectors,particularly for in vivo therapy, for example, in somatic cell therapyor direct in vivo applications, many other applications exist such as inin vitro assays.

Various vectors have characteristics that make them desirable forcertain applications. For example, a retroviral vector can be used toinfect a host cell and have the genetic material integrated into thathost cell with high efficiency. One example of such a vector is amodified Moloney murine leukemia virus (MMLV), which has had itspackaging sequences deleted to prevent packaging of the entireretroviral genome. However, that retrovirus does not transduce restingcells. Additionally, since many retroviruses typically enter cells viaspecific receptors, if the specific receptors are not present on a cellor are not present in large enough numbers, the infection is either notpossible or is inefficient. Concerns have also been expressed as aresult of outbreaks of wild-type viruses from the recombinant MMLVproducing cell lines, i.e., reversions.

An adenovirus vector can infect a wide range of cells. However, thetransferred genetic material is not integrated. Therefore, it must beadministered repeatedly. Additionally, most individuals have naturalimmunity to such vectors. Thus, high volumes of virus are needed whenusing the vector for gene delivery. The reason for this is the body'sown immune system more readily attacks these vectors.

Recently, attention has focused on lentiviral vectors such as thosebased upon the primate lentiviruses, e.g., human immunodeficiencyviruses (HIV) and simian immunodeficiency virus (SIV). HIV vectors caninfect quiescent cells in addition to dividing cells. Moreover, by usinga pseudotyped vector (i.e., one where an envelope protein from adifferent species is used), problems encountered with infecting a widerange of cell types can be overcome by selecting a particular envelopeprotein based upon the cell you want to infect. Additionally, in view ofthe complex gene splicing patterns seen in a lentiviruses such as HIV,multivalent vectors (i.e., those expressing multiple genes) having alentiviral core, such as an HIV core, are expected to be more efficient.Yet, despite the advantages that HIV based vectors offer, there is stilla concern with the use of HIV vectors in view of the severity of HIVinfection. Thus, means for providing additional safeguards, such asattenuated forms that are less likely to revert to a wild type virus aredesirable.

Variations can be made where multiple modifications are made, such asdeleting nef. rev, vif and vpr genes. One can also have the 3′ and 5′ U3deleted LTRs.

Marasco et al. discovered a method by which one could express antibodieswithin a cell and have them bind to a target within that cell. [See U.S.Pat. No. 5,851,829 to Marasco and Haseltine]. These intracellularlyexpressed antibodies (intrabodies) can be used in a method of functionalgenomics. In this manner, one can take a specific unknown gene, expressits gene product, use that gene product to generate an antibody theretoand use the antibody intracellularly to “knock-out” the putative proteinin the cell. Thereafter one can compare that cell to a control cell todetermine the effect the loss of its gene product has on the cell inboth in vitro and in vivo systems. This method requires generation of aspecific antigen and antibody thereto. It would be desirable to have amethod to take advantage of the efficiencies of this approach with largenumbers of members of a particular group.

It would be highly desirable to have a vector and a method of usethereof where one could look for any molecule resulting in a particularfunction and rapidly determine that molecule, e.g. protein. It would bevery desirable to be able to do this in an automated manner permittingrapid identification of the desired molecule.

SUMMARY OF THE INVENTION

We have now discovered a plurality of vectors, wherein the group ofvectors can be used for gene delivery. The vectors preferably contain aheterologous nucleic acid sequence, preferably encoding proteins such asantibodies, angiogenic proteins, growth factors, receptors, cytokines,antiangiogenic proteins, co-stimulatory proteins, peptides, ribozymesand antisense molecules. Preferably the heterologous nucleic acidsequences are genes encoding proteins such as antibodies or angiogenicproteins. More preferably the nucleic acid sequences are operably linkedto an inducible promoter. The vectors can be used to transduce aplurality of cells. Preferably, the vectors contains a marker gene topermit rapid identification and selection of transformed cells.Thereafter, those cells are screened to identify a cell exhibiting adesired phenotype. Cells exhibiting a desired phenotype are selected andthe particular target molecule resulting in the phenotype identified.

In one preferred embodiment the plurality of vectors are lentiviralvectors. These lentiviral vectors preferably contain a selectablemarker.

The lentivirus vectors include, for example, human immunodeficiencyvirus (HIV) (e.g. HIV-1 and HIV-2), feline immunodeficiency virus (FIV),or visna virus. A vector containing such a lentivirus core (e.g. gag)can transduce both dividing and non-dividing cells.

The lentiviral virion (particle) is expressed by a vector systemencoding the necessary viral proteins to produce a virion (viralparticle). Preferably, there is at least one vector containing a nucleicacid sequence encoding the lentiviral pol proteins necessary for reversetranscription and integration, operably linked to a promoter.Preferably, the pol proteins are expressed by multiple vectors. There isalso a vector containing a nucleic acid sequence encoding the lentiviralgag proteins necessary for forming a viral capsid operably linked to apromoter. In one embodiment, the gag-pol genes are on the same vector.Preferably, the gag nucleic acid sequence is on a separate vector thanat least some of the pol nucleic acid sequence, still more preferably itis on a separate vector from all the pol nucleic acid sequences thatencode pol proteins.

In one embodiment, the gag sequence does not express a functional MAprotein, i.e. the vector can still transduce cells in the absence of theentire MA or a portion thereof, if a myristylation anchor is provided.This can be accomplished by inactivating the “gene” encoding the MA byadditions, substitutions or deletions of the MA coding region.Preferably, this is done by deletion. Preferably, at least 25% of the MAcoding region is deleted, more preferably, at least 50% is deleted,still more preferably, at least 60%. even more preferably at least 75%,still more preferably, at least 90%, yet more preferably at least 95%and most preferably the entire coding region is deleted. However. inthat embodiment, a myristylation anchor (sequence) is still required.Preferably, the myristylation sequence is a heterologous (i.e.,non-lentiviral) sequence.

In another embodiment the lentiviral vector is another form ofself-inactivating (SIN) vector as a result of a deletion in the 3′ longterminal repeat region (LTR). Preferably, the vector contains a deletionwithin the viral promoter. The LTR of lentiviruses such as the HIV LTRcontains a viral promoter. Although this promoter is relativelyinefficient, when transactivated by e.g. tat, the promoter is efficientbecause tat-mediated transduction increases the rate of transcriptionabout 100 fold. However, the presence of the viral promoter caninterfere with heterologous promoters operably linked to a transgene. Tominimize such interference and better regulate the expression oftransgenes, the lentiviral promoter is preferably deleted.

Preferably, the vector contains a deletion within the viral promoter.The viral promoter is in the U3 region of the 3′ LTR. A preferreddeletion is one that is 120 base pairs between Scal and PvuI sites, e.g.corresponding to nucleotides 9398-9518 of HIV-1 proviral clone HXO2,encompassing the essential core elements of the HIV-1 LTR promoter (TATAbox, SP1 and NF—Pb binding sites). After reverse transcription, thedeletion is transferred to the 5′ LTR, yielding a vector/provirus thatis incapable of synthesizing vector transcripts from the 5′ LTR in thenext round of replication. Thus, the vector of the present inventioncontains no mechanism by which the virus can replicate as it cannotexpress the viral proteins.

In another embodiment the vector is a tat deleted vector. This can beaccomplished by inactivating at least the first exon of tat by knowntechniques such as deleting it or inserting a stop codon. Alternatively,one can extend the U3 LTR deletion into the R region to remove the TARelement.

Variations can be made where the lentiviral vector has multiplemodifications as compared to a wildtype lentivirus. For example, withHIV being nef-, rev-, vpu-, vif- and vpr-. In addition one can haveMA-gag, 3′ and 5′ U3 deleted LTR and variations thereof.

The vector(s) do not contain nucleotides from the lentiviral genome thatpackage lentiviral RNA, referred to as the lentiviral packagingsequence. In HIV this region corresponds to the region between the 5′major splice donor and the gag gene initiation codon (nucleotides301-319).

The env, gag and pol vector(s) forming the particle preferably do notcontain a nucleic acid sequence from the lentiviral genome thatexpresses an envelope protein. Preferably, a separate vector thatcontains a nucleic acid sequence encoding an envelope protein operablylinked to a promoter is used. This env vector also does not contain alentiviral packaging sequence. In one embodiment the env nucleic acidsequence encodes a lentiviral envelope protein.

In another embodiment the envelope protein is not from the lentivirus,but from a different virus. The resultant particle is referred to as apseudotyped particle. By appropriate selection of envelopes one can“infect” virtually any cell. Thus, the vector can readily be targeted toa specific cell. In Table A, a list of viruses and cell types that theypreferentially infect is provided. Depending upon the host cell that onedesires to target, one would take the envelope glycoprotein of thatvirus and use it as the envelope protein of the pseudotype vector. Inanother example, one can use an env gene that encodes an envelopeprotein that targets an endocytic compartment such as that of theinfluenza virus, VSV-G, alpha viruses (Semliki forest virus, Sindbisvirus), arenaviruses (lymphocytic choriomeningitis virus), flaviviruses(tick-borne encephalitis virus, Dengue virus), rhabdoviruses (vesicularstomatitis virus, rabies virus), and orthomyxoviruses (influenza virus).In another embodiment, filoviruses are specific for vascular endotheliumcells. Therefore, using an envelope glycoprotein such as GP₁, GP₂ fromEbola, one can selectively target vascular endothelium cells. When thedesired host cell is a brain cell, the envelope glycoprotein fromvaricella zoster virus (VZV) can be used.

The envelope protein used can be a truncated protein as long as itretains the receptor binding site, which permit viral entry to a cell.Thus, for example, the cytoplasmic tail and/or transmembrane domains canbe deleted.

The preferred lentivirus is a primate lentivirus [U.S. Pat. No.5,665,577] or a feline immunodeficiency virus (FIV) [Poeschla, E. M., etal., Nat. Medicine 4:354-357 (1998)]. The pol/gag nucleic acidsegment(s) and the env nucleic acid segment will when expressed producean empty lentiviral particle. By making the above-describedmodifications such as deleting the tat coding region the MA codingregion, or the U3 region of the LTR, the possibility of a reversion to awild type virus has been reduced.

A desired nucleic acid segment (sometimes referred to as the targetmolecule) such as a family of nucleic acid sequences can be insertedinto the empty lentiviral particles by use of a plurality of vectorseach containing a nucleic acid segment of interest and a lentiviralpackaging sequence necessary to package lentiviral RNA into thelentiviral particles (the packaging vector). Preferably, the packagingvector contains a 5′ and 3′ lentiviral LTR with the desired nucleic acidsegment inserted between them. The nucleic acid segment can be antisensemolecules or more preferably, encodes a protein such as an antibody. Thepackaging vector preferably contains a selectable marker. These are wellknown in the art and include genes that change the sensitivity of a cellto a stimulus such as a nutrient, an antibiotic, etc. Genes includethose for neo, puro, tk, multiple drug resistance (MDR), etc. Othergenes express proteins that can readily be screened for such as greenfluorescent protein (GFP), blue fluorescent protein (BFP), luciferase,LacZ, nerve growth factor receptor (NGFR), etc.

When an inducible promoter is used with the target molecule minimalselection pressure is exerted on the transformed cells for those cellswhere the target molecule is “silenced”. Thus, identification of cellsdisplaying the marker also identifies cells that can express the targetmolecule. If an inducible promoter is not used, it is preferable to usea “forced-expression” system where the target molecule is linked to theselectable marker by use of an internal ribosome entry site (IRES) [seeMarasco et al., PCT/US96/16531].

IRES sequences are known in the art and include those fromencephalomyocarditis virus (EMCV) [Ghattas, I. R. et al., Mol CellBiol., 11: 5848-5849 (1991)]; BiP protein [Macejak and Sarnow, Nature,353:91 (1991)]; the Antennapedia gene of Drosophila (exons d and e) [Ohet al., Genes & Dev., 6: 1643-1653 (1992)]; those in polio virus[Pelletier and Sonenberg, Nature 334:320325 (1988); see also Mountfordand Smith, TIG, 11:179-184 (1985)]. Preferably, the target molecule isoperably linked to an inducible promoter. Such systems allow the carefulregulation of gene expression. See Miller, N. and Whelan, J., Human GeneTherapy, 8: 803-815 (1997). Such systems include those using the lacrepressor from E. coli as a transcription modulator to regulatetranscription from lac operator-bearing mammalian cell promoters [Brown,M. et al., Cell, 49:603-612 (1987)] and those using the tetracyclinerepressor (tetR) [Gossen. M., and Bujard, H., Proc. Natl. Acad Sci. USA89:5547-5551 (1992); Yao. F. et al., Human Gene Therapy, 9:1939-1950(1998); Shockelt, P., et al., Proc. Natl. Acad Sci. USA., 92:6522-6526(1995)]. Other systems include FK506 dimer VP16 or p65 using astradiol.RU486. diphenol murislerone or rapamycin [see Miller and Whelan, supraat FIG. 2]. Inducible systems are available from Invitrogen, Clontechand Arid. Systems using a repressor with the operon are preferred.Regulation of transgene expression in target cells represents a criticalaspect of gene therapy. For example, a lac repressor combined thetetracycline repressor (tetR) with the transcription activator (VP16) tocreate a tetR-mammalian cell transcription activator fusion protein, tTa(tetR-VP 16), with the tetO-bearing minimal promoter derived from thehuman cytomegalovirus (HCMV) major immediate-early promoter to create atetR-tet operator system to control gene expression in mammalian cells.Recently Yao and colleagues [F. Yao et al., Human Gene Therapy, supra]demonstrated that the tetracycline repressor (tetR) alone, rather thanthe tetR-mammalian cell transcription factor fusion derivatives canfunction as potent trans-modulator to regulate gene expression inmammalian cells when the tetracycline operator is properly positioneddownstream for the TATA element of the CMVIE promoter. One particularadvantage of this tetracycline inducible switch is that it does notrequire the use of a tetracycline repressor-mammalian cellstransactivator or repressor fusion protein, which in some instances canbe toxic to cells [M. Gossen et al., Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992); P. Shockett et al., Proc. Natl. Acad. Sci. USA,92:6522-6526 (1995)], to achieve its regulatable effects. Preferably,the repressor is linked to the target molecule by an IRES sequence.Preferably, the inducible system is a tetR system. More preferably thesystem has the tetracycline operator downstream of a promoter's TATAelement such as with the CMVIE promoter. See FIG. 4.

The target molecules used in one embodiment have genes encodingantibodies intended to be expressed intracellularly. Antibodies havelong been used in biomedical science as in vitro tools for theidentification, purification and functional manipulation of targetantigens. Antibodies have been exploited in vivo for diagnostic andtherapeutic applications as well. Recent advances in antibodyengineering have now allowed the gene encoding antibodies to bemanipulated so that the antigen binding domain can be expressedintracellularly. The specific and high-affinity binding properties ofantibodies, combined with the creation of large human immunoglobulinlibraries and their ability to be stably expressed in preciseintracellular locations inside mammalian cells, has provided a powerfulnew family of molecules for gene therapy applications. Theseintracellular antibodies are termed “intrabodies ” [W. Marasco et al.,Gene Therapy, 4:11-15 (1997)]. Preferably, the genes encode a singlechain antibody. The molecules preferably contain a tag such as HA sothat the molecule can be identified later.

The antibodies are preferably obtained from a library of antibodies suchas a phage display library.

Thereafter the lentiviral vectors are used to transduce a host cell. Onecan rapidly select the transduced cells by screening for the marker.Thereafter, one can take the transduced cells and grow them under theappropriate conditions or insert those cells e.g. spleen cells or germcells, into a host animal.

The promoter is induced and then one screens for cells and/or animalsdisplaying a particular phenotype. Using the tag contained on themolecule, e.g. antibody, one can obtain the molecules, e.g., antibodythat resulted in the desired phenotype. In one example, the antibody canthen be used identify the antigen it bound to, if that is desired.

This method permits one to use a multitude of molecules to identify aspecific molecule providing the desired function from a large group ofmolecules without first needing to know the specific identity of anymember.

In another embodiment, the nucleic acid sequence encodes an angiogenesispromoting compound and is delivered selectively to vascular endothelialcells. To selectively target such cells, a pseudotyped lentiviralparticle is used. The envelope is from a filovirus such as Ebola.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a schematic of a lentiviral vector system.

FIGS. 2A and 2B show tetR-mediated repression of transcriptioninitiation. FIG. 2A outline of the single inducible cassette and theexpected polycistronic mRNA. FIG. 2B shows a Northern blot analysis.Mock-treated Vero cells and cells transfected independently with theempty vector, the pcDNAtetR plasmid, the one piece control (1Pc) and theone piece inducible (1Pi) were harvested after 2 days posttransfectionand total RNA was separated using the TRizol reagent, followed bychloroform extraction and precipitation with isopropanol. Total RNA (20μg) was run in denaturing conditions and blotted on Hybond-N membranesto detect the presence of specific mRNAs that hybridize with aradiolabeled tetR probe (XbaI-EcoRI DNA fragment indicated in 2A). Atranscript of about 0.6 Kb corresponding to the tetR mRNA is shown.Tetracycline regulation of the bicistronic mRNA expression from theinducible cassette is observed.

FIG. 3 represents the results obtained from transfecting with thetetracycline-inducible plasmids at various concentrations (2. 5 or 10∥g).

FIG. 4 is a schematic diagram of a cloning vector, pGEM-72f(t) used totransfect the library of target molecules.

FIGS. 5A and B are a schematic of an HIV-1 based retroviral vectorincluding a packaging vector, HIVNtetOIR, (FIG. 5A) and the packingdefective lentiviral vectors (FIG. 5B).

FIG. 6 is a schematic showing how to isolate single-chain antibodies(sFv) by phage display technology.

FIG. 7 is a comparison of the regulation of hEGF expression using twoseparate plasmids or a single control cassette. Vero cells in duplicate,were independently transfected with the 2 plasmid system using 0.5 μg ofpCMVtetOhEGF (2Pi) or the non-regulated version pCMVhEGF (2Pc) alone(white bars), or in combination with 2 μg of pcDNAtetR (striped andblack bars) or empty vector pcDNA 3.1 (−), either in the absence (whiteand striped bars) or presence of 1 μg/ml of tetracycline (black bars).To test the one piece control (1Pc) and inducible plasmids (1Pi), cellsin triplicates were independently transfected with 2.5 μg of thecorresponding DNA in the absence (striped bars) or presence (black bars)of the antibiotic. Extracellular medium was collected from thetransfected cells at the indicated times and the expression of HEGF wasmeasured by ELISA.

FIG. 8 shows dose-response effects to tetracycline. Vero cellstransfected with the 1Pi cassette were treated and grown in the presenceof increasing concentration of tetracycline in the culture media. After48 hr., the amount of HEGF released to the medium was analyzed by ELISA.

FIG. 9 shows the reversible effects of our single cassette in VEROcells. Transfected cells were cultured in the absence (white bars) orpresence of tetracycline during the entire experiment (black bars) oralternatively, after 24 hr. treatment, the cells were maintained inmedia without the inducer (shaded bars). Culture media was analyzed forhEGF production at the indicated time points.

FIG. 10 shows regulation of eGFP expression in different cell lines.Non-transfected and cells transfected either with an empty vector,pcDNA3.1 (−) or with our 1Pc or 1Pi plasmids were analyzed by FACSanalysis after 48 hr. posttransfection, to determine endogenous eGFPexpression in different cell lines in the absence (striped bars) orpresence (black bars) of 1 μg/ml tetracycline.

FIG. 11A-H show co-expression of eGFP and tetR in transfected Verocells. Vero cells transfected with 1Pc (FIGS. 11A-D) or 1Pi (FIGS.11E-H) were grown for two days in the absence (FIGS. 11A, 11B, 11E and11F) or presence (FIGS. 11C, 11D, 11G and 11H) of the inducer prior toanalysis. Simultaneous observation of eGFP (FIGS. 11A, 11C, 11E and 11G)and tetR (FIGS. 11B, 11D, 11F and 11H) expression was performed byimmuno-reaction of the tetR protein using a primary antibody againsttetR and a secondary goat anti-mouse IgG coupled to PE that allowsdetection of the immune-complexes at different wavelengths.

FIG. 12 shows tetR-mediated repression is enhanced by inserting a NLSsequence. Vero cells transfected either with the control (1Pc or1Pc.NLS) or the inducible (1Pi or 1Pi.NLS) version of our constructswere grow in the absence (striped bars) or presence (black bars) oftetracycline. Aliquots of harvested supernatants were analyzed todetermine the amount of hEGF secreted into the culture media.

FIGS. 13A-D show immunolocalization of tetR after addition of the NLSsequence. Localization of tetR protein after transfection of VERO cellswith different plasmid constructs was performed by conventionalimmunofluorescence. Cells transfected with a control plasmid (FIG. 13A),the pcDNAtetR plasmid (FIG. 13B), and the 1Pi (FIG. 13C) or1Pi.NLS.(FIG. 13D) in the presence of tetracycline were fixed with 4%formaldehyde/PBS and permeabilized with a detergent before incubationwith a monoclonal antibody against tetR. After 2 hr. incubation with theprimary antibody, a goat anti-mouse IgG coupled to FITC allowedvisualization under a fluorescence microscope (Final magnification400×).

FIG. 14 shows a comparison of infection of monkey CD8-(striped bars) andhuman CD8-(solid black bars) PBMC's infected by two pseudotyped primatelentivirus (HIV and SHIV)

DETAILED DESCRIPTION OF THE INVENTION

We have now discovered anew vector system using a plurality of vectors,wherein the group of vectors encode a lentiviral particle that can beused for gene delivery. Preferably, the lentiviral particles arepseudotyped having an envelope protein that differs from the lentiviralcapsid. These particles can be used to transfer nucleic acid sequences.The molecules can be proteins such as antibodies, growth factors,angiogenic modulating proteins, receptors and cytokines, peptides, andantisense molecules. In one embodiment, the nucleic acid sequences aregenes encoding proteins such as antibodies, or angiogenic promotingmolecules. More preferably the proteins are operably linked to aninducible promoter. The vectors can be used to transduce a plurality ofcells. In one alternative, the vectors contain a marker gene to permitrapid identification and selection of transformed cells. Thereafter,those cells are screened to identify a cell exhibiting a desiredphenotype. Cells exhibiting a desired phenotype are selected and theparticular target molecule resulting in a specific phenotype areidentified.

In one preferred embodiment the plurality of vectors are lentiviralvectors. These lentiviral vectors preferably contain a selectablemarker.

The lentivirus vectors include, for example, human immunodeficiencyvirus (HIV), feline immunodeficiency virus (FIV), or visna virus. Avector containing such a lentivirus core (e.g. gag gene) can transduceboth dividing and non-dividing cells.

The preintegration complex of lentiviruses, a family of retroviruseswhich includes the human immunodeficiency virus type 1 (HIV-1), havebeen shown to possess nuclear targeting signals which allow theseviruses to infect non-dividing cells including macrophages. The capacityof HIV-1 [P. Lewis et al., EMBO J., 11 :3053-3058 (1992); M. Burinsky etal., Proc. Natl. Acad. Sci. USA, 89:6580-6584 (1992)] vectors to stablytransduce non-dividing cells has been demonstrated in vitro [J. Reiseret al., Proc. Natl. Acad. Sci. USA, 93:15266-15271 (1996)] and also invivo [L. Naldini et al., Science, 272:263-267 (1996)]. Thus, thesevectors are capable of long-term expression.

A second feature of HIV-1 based vectors is the ability to manipulate thetarget cell range by substituting the HIV-1 envelope glycoprotein,gp160, with envelope proteins from other viruses which confer anextended host range that can be specifically targeted. The resultantparticle is referred to as a pseudotyped particle. For example, robustassociation between the G protein of vesicular stomatitic virus (VSV)-Gprotein and the HIV-1 virion core allows virus particles to beconcentrated without loss of infectivity and has enabled the productionof HIV-1 vector stocks with titers of about 10⁹/ml [J. Reiser et al.,Proc. Natl Acad. Sci. USA, 93:15266-15271 (1996); R. Akkina et al., J.Virol., 70:2581-2585 (1996); J. Yee et al., Proc. Natl. Acad. Sci. USA,91:9564-9568 (1994)]. Lentiviral vectors such as HIV-1 vectors havetherefore been developed to a point of clinical utility and offerconsiderable potential as an in vivo tool for the manipulation of bothdividing and non-dividing cells.

By appropriate selection of envelopes one can selectively “infect”virtually any cell. Table A provides a list of different viruses andcells that they infect. Using this information, one can readily choose aviral envelope protein to pseudotype in the present invention.

For example, filoviruses such as Ebola selectively infect vascularendothelial cells. These cells are involved in the circulatory system.Current protocols where circulatory problems exist involve administeringan angiogenic protein such as vascular endothelial growth factor (VEGF)or basicFGF to adjoining cells to promote the growth of new blood cells.One method is to administer a gene encoding a protein such as anangiogenic protein, where the protein preferably has a secretorysequence. These genes are typically transferred to adjoining cellsbecause it is difficult to transform the vascular endothelial cells.However, even where the method involves a gene delivery system such as acatheter, the transformation and expression of the angiogenic protein isnot selective. While the individual having circulatory problems benefitsby the expression of the angiogenic protein with respect to thecirculatory problem, e.g., ischemia, that individual can be at increasedrisk from the undesired angiogenesis. One model of tumor growth suggeststhat it is the development of blood vessels near tumors that permit thetumor to grow and/or metastize. Thus, precise control over the targetingof cells and the expression of the protein is extremely important. AnEbola pseudotyped lentivirus provides such specific targeting, whereasthe use of an inducible promoter as described later permits precision inexpression.

In another example, one can use an env gene that encodes an envelopeprotein that targets an endocytic compartment such as that of theinfluenza virus, VSV-G, alpha viruses (Semliki forest virus, Sindbisvirus), arenaviruses (lymphocytic choriomeningitis virus), flaviviruses(tick-borne encephalitis virus, Dengue virus), rhabdoviruses (vesicularstomatitis virus, rabies virus), and orthomyxoviruses (influenza virus).

Other envelopes that can preferably be used include those from MoloneyLeukemia Virus such as MLV-E, MLV-A and GALV. These latter envelopes areparticularly preferred where the host cell is a primary cell.

Other envelope proteins can be selected depending upon the desired hostcell. For example, targeting specific receptors such as dopaminereceptor for brain delivery. VZV will infect brain cells, ganglion cellsand astrocytes. TABLE A HSV Human diploid fibroblast, primary humanembryonic kidney and primary rabbit kidney; Hep 2 Hela VZV Diploidfibroblasts, primary human kidney and primary monkey kidney CMV Humandiploid fibroblasts. Influenza Primary monkey kidney cells; Madin -Darby kidney (MDCK) Paranfluenza Primary monkey kidney, human embryonickidney cells Mumps Primary rhesus monkey kidney cells RSV Hep 2 cellsFiloviruses Vascular endothelial Ad Human embryonic kidney (Hep 2)Rhinoviruses Human embryonic diploid Fb lines Measles Primary monkeykidney and primary human embryonic kidney cells Rubella 1° African green(vervet) monkey kidney cells HIV Macrophages, lymphocytes ArbovirusEmbryonated eggs, chick embryo fibroblasts, some insect lines II. INDEPTH Enteroviruses Eg Polio, Coxsackie, Echoviruses Human TC = WI-38,HEK Monkey kidney tissue culture RD cell line (human rhabdomyosarcoma)HeLa Buffalo green monkey, primary human kidney cells Human fetaldiploid kidney cells Poliovirus Primary monkey kidney cells, diploidFibroblasts, Hep-2, HeLa, Vero Coxsackie B, A & Echovirus CBV = Primarykidney cells from monkeys or human embryos; Hep-2, HeLa Vero CAV =inoculation of newborn mice Echo = Primary monkey and human embryokidney; human diploid fibroblast cell lines Rhinoviruses Rhesus monkeykidney cell lines. Human embryonic kidney cells; human amnion; diploidfibroblasts from human embryonic lung, tonsil, liver, intestine andskin. adult fibroblast cell lines from aorta and gingiva L132, KB,Hep-2, and HeLa (transformed cells); WI-38 (diploid Fibroblast), fetaltonsil line (Cooney strain); MRC-5 line Hepatitis HAV Primary explantcultures of marmoset liver Fetal rhesus monkey kidney cell line (FRhK6)Primary African green monkey kidney cells (AGMK), Primary humanfibroblast cells. Transformed human diploid lung cells (MRC5) Cell linesderived from: (I) AGMK = BS-C-1, Vero, BGMK fetal rhesus kidney = FRhK4,FRhK6, Frp/3 Human hepatona = PLC, PRf/5 HBV 1° cultures of normal adultor fetal human hepatocytes tissue = liver Hepatitis E Diploid humanembryonic lung cells, 1° cynomolgus monkey hepatocyte culture Host rangedepends on several genes Norwalk Not propagated in cell culture or inhuman embryonic intestinal organ culture Astroviruses Tertiary bovineembryo kidney cells (BEK) Primary neonatal bovine kidney cells (NBK)Togavirus Chicken embryo fibroblasts (CEF) Baby hamster kidney cells(BHK) Alphaviruses Avian embryo cells Continuous cell lines = babyhamster kidney (BHK-21); monkey kidney (Vero) and mosquito (C6/36)Rubella Rabbit kidney line RK-13, AGMK, Vero, BHK-21 Baby hamster kidneyFlavivirus primary chick or duck embryonic cells. Grow in continuouscell cultures from human, monkey, rodent, swine avian, amphibian andanthropod. HeLa, BHK-21, porcine kidney (PS), SW-13 (human adrenalcarcinoma), Aedes mosquito cells Filoviruses Ebola Tropic for vascularendothelial cells Pestiviruses Can be propagated in cultures of primaryand permanent cells of their host species eg. deer, african buffalo,giraffe, wildebeest, most pigs, cattle, sheep, goats Coronavirus None ofhuman coronavirus grow well in cell culture without extensive adaptationby passage. Some grown in human embryonic tracheal organ culture,primary or secondary human fibroblast cell lines (HEK). Most sensitiveis diploid intestinal cell line MA-177. Fetal tonsil diploid cells (FT)Rhadovirus BHK-21, Vero, primary chick embryo cells, Chinese hamsterovary cells, fish cell lines. Filoviridae Passaged in Monkeys, guineapigs, suckling mice and hamsters. Cell culture: vero cells (clone E-6),MA-108, SW-13, NIH-3T3, BSC-1 MDCK HeLa. Paranfluenza Embryonatedchicken eggs, monkey kidney cells. Mumps Virus Primary rhesus monkeykidney cells, BSC-1, Vero, MDBK, HeLa Measles Virus Peripheral bloodleukocytes or respiratory secretions inoculated onto primary human ormonkey kidney cells; human cord blood leukocytes. Cell cultures: Vero,CV-1 (continuous monkey kidney cell line) EBV transformed marmoset Blymphocyte line; B95-8; A549 cells Respiratory Syncytial Virus (RSV)Hep-2, HeLa, wide variety of human and animal cells Orthomyxvirus eg.Influenza MDCK, primary cell cultures including monkey kidney, calfkidney, hamster kidney and chicken kidney Bunyaviridae Most cellcultures Arenaviridae eg LCMV Most cell types from many different hostspecies but not lymphocytes and terminally differentiated neuronsReoviridue MDCK, 3T3 (primary fibroblasts) Reoviruses Mouse L929fibroblasts, Madin-Darby bovine kidney (MDBK) rhesus monkey kidney(LLC-MK2) human embryonic intestinal cells Rotaviruses Bovine RV =Bovine kidney cells, BSC-1, polarized human intestinal epithelial(CaCO-2) Rhesus RV = MDCK, CaCO-2 HTLV T cells, human cord or PBL's,EBV-transformed B cell lines, B cells, immature cells from bone marrowwhich do not have T-cell phenotype; macrophages, cells of neural originGrowth in vitro (I) HTLV-I = CD4⁺CD8⁻ (ii) HTLV-II = CD8⁺ PolyomavirusesVero cells; epithelial and fibroblastic cells of human origin, HEK,diploid lung fibroblasts (WI38), urothelial cells, primary human fetalglial (PHFG) Human fetal Schwann cells (glial lineage), human fetalastrocytes, HEK Papillonaviruses Not successfully propagated inmonolayer cell culture to yield virus particles Adenovirus Best grown inhuman embryonic kidney cells (HEK) A549 - derived from a human lungcarcinoma Hep-2, HeLa, KB, monkey kidney cells lines eg. Vero 293 cellline - primary HEK transformed by Ad5 (EIA&B positive) cells primarymonkey kidney cells Parvovirus Human erythroid cells and cultures ofhuman bone marrow, peripheral blood leukocytes, immuophenotype of cellsinfected = surface expression of glycophorins, HLA, leucosialin (CD43)andplatelet glycoprotein IV (CD36) HSV Vero, Hep-2, baby hamster kidneycells EBV B cells, EBV positive malignant cells BL (Burkitt cells) Invivo = permissive in pharyngeal epithelium. Immunophenotype of BL cells:BL group 1 = CD10, CD77 BL group III CD23, CD30, CD39, CD70, B7, LFA3,ICAM1 CMV Found in virtually all organ systems. Varicella Zoster virusVZV found in thyroid, human melanoma, human embryo lung fibroblast etc.Grows in primary diploid and continuous human cell cultures such asprimary human foreskin fibroblasts, kidney, thyroid, amnion cells,primary human keratinocytes, adult brain cells, ganglion cells,astrocytes, Schwanncells VZV can be propagated in W1-38 or MRC-5 cellsInfects a human neuroblastoma cell line (IMR-32) Replicates inEBV-transformed human B lymphocytes in vitro Infects activated T cellsin vitro Grows in primary rhesus monkey kidney cells, African greenmonkey kidney cells, guinea pig embryo fibroblasts. Primate cell lines -BSC-1 and Vero Human Herpesvirus 6 Cell tropism = CD4⁺ T cells in vitro= primary human monoyte/macrophage, NK cells, megakaryocytes, glial celllineages and transformed cervical epithelial cells, Molt-3 (T-cellline), MT-6 (HTLV-1⁺ human T cell lymphoma line) HHV7 CD4⁺ T cellsPoxvirus Epithelial cells. Vaccinia - broad host range Small pox -macrophages, epithelial and mucous membranes

In one embodiment one uses the entire envelope glycoprotein, but onedoes not have to. Envelope glycoproteins are typically processed from asingle precursor to two proteins. For example, with HIV, the gp160precursor is processed to form a gp120 and gp4l. Similarly, Ebola has aprecursor GP which by post-transcriptional modification becomes a GP₁and a GP₂. In some instances one can enhance processing by removing thecleavage site, transmembrane domain and/or cytoplasmic tail. Otherportions can be deleted also. The key is to retain the cellular bindingportion. Typically this is the receptor binding portion(s). Thus, if thevirus targets one receptor, only that receptor binding site may benecessary. However, there are instances where co-factors are also usedfor viral entry and those sites also need to be retained.

In an alternative embodiment, one can use different lentiviral capsidswith a pseudotyped envelope. For example, FIV or SHIV [U.S. Pat. No.5,654,195] a SHIV pseudotyped vector can readily be used in animalmodels such as monkeys.

The preferred lentivirus is a primate lentivirus [U.S. Pat. No.5,665,577] or a feline immunodeficiency virus (FIV) [Poeschla, E. M., etal., Nat. Medicine 4:354-357 (1998)] The pol/gag nucleic acid segment(s)and the env nucleic acid segment will when expressed produce an emptylentiviral particle. By making the above-described modifications such asdeleting the tat coding region, the MA coding region, or the U3 regionof the LTR, the possibility of a reversion to a wild type virus has beenreduced to virtually nil.

The lentiviral virion (particle) is expressed by a vector systemencoding the necessary viral proteins to produce a virion (viralparticle). Preferably, there is at least one vector containing a nucleicacid sequence encoding the lentiviral pol proteins necessary for reversetranscription and integration, operably linked to a promoter.Preferably, the pol proteins are expressed by multiple vectors. There isalso a vector containing a nucleic acid sequence encoding the lentiviralgag proteins necessary for forming a viral capsid operably linked to apromoter. Preferably, this gag nucleic acid sequence is on a separatevector thari at least some of the pol nucleic acid sequence, still morepreferably it is on a separate vector from all the pol nucleic acidsequences that encode pol proteins.

Numerous modifications can be made to the vectors, which are used tocreate the particles to further minimize the chance of obtaining wildtype revertants. These include deletions of the U3 region of the LTR,tat deletions and matrix (MA) deletions.

The gag, pol and env vector(s) do not contain nucleotides from thelentiviral genome that package lentiviral RNA, referred to as thelentiviral packaging sequence. In HIV this region corresponds to theregion between the 5′ major splice donor and the gag gene initiationcodon (nucleotides 301-319).

The vector(s) forming the particle preferably do not contain a nucleicacid sequence from the lentiviral genome that expresses an envelopeprotein. Preferably, a separate vector that contains a nucleic acidsequence encoding an envelope protein operably linked to a promoter isused. This env vector also does not contain a lentiviral packagingsequence. In one embodiment the env nucleic acid sequence encodes alentiviral envelope protein.

A desired family of heterologous nucleic acid segments (sometimesreferred to as the target molecules) can be inserted into the emptylentiviral particles by use of a plurality of vectors each containing anucleic acid segment of interest and a lentiviral packaging sequencenecessary to package lentiviral RNA into the lentiviral particles (thepackaging vector). Preferably, the packaging vector contains a 5′ and 3′lentiviral LTR with the desired nucleic acid segment inserted betweenthem. The nucleic acid segment can be an antisense molecule or morepreferably, encodes a protein such as an antibody. The packaging vectorpreferably contains a selectable marker. These are well known in the artand include genes that change the sensitivity of a cell to a stimulussuch as a nutrient, an antibiotic, etc. Genes include those for neopuro, tk, multiple drug resistance (MDR), etc. Other genes expressproteins that can readily be screened for such as green fluorescentprotein (GFP), blue fluorescent protein (BFP), luciferase, LacZ, nervegrowth factor receptor (NGFR), etc.

As used herein, the introduction of DNA into a host cell is referred toas transduction, sometimes also known as transfection or infection.

One can set up systems to screen cells automatically for the marker. Inthis way one can rapidly select transduced cells from non-transformedcells. For example, the resultant particles can be contacted with aboutone million cells. Even at transduction rates of 10-15% one will obtain100-150,000 cells. An automatic sorter that screens and selects cellsdisplaying the marker, e.g. GFP, can be used in the present method.

When an inducible promoter is used with the target molecule, minimalselection pressure is exerted on the transformed cells for those cellswhere the target molecule is “silenced”. Thus, identification of cellsdisplaying the marker also identifies cells that can express the targetmolecule. If an inducible promoter is not used, it is preferable to usea “forced-expression” system where the target molecule is linked to theselectable marker by use of an internal ribosome entry site (IRES) (seeMarasco et al., PCT/US96/16531). In this manner, virtually all cellsselected on the basis of the marker also contain and can express thetarget molecule.

IRES sequences are known in the art and include those fromencephalomycarditis virus (EMCV) [Ghattas, I. R. et al., Mol. Cell.Biol., 11:5848-5849 (1991)]; BiP protein [Macejak and Sarnow, Nature,353:91 (1991)]; the Antennapedia gene of Drosophila (exons d and e) [Ohet al., Genes & Development, 6:1643-1653 (1992)]; those in polio virus[Pelletier and Sonenberg, Nature, 334:320-325 (1988); see also Mountfordand Smith, TIG, 11: 179-184 (1985)].

Preferably, the target molecule is operably linked to an induciblepromoter. Such systems allow the careful regulation of gene expression.See Miller, N. and Whelan, J., Human Gene Therapy, 8:803-815 (1997).Such systems include those using the lac repressor from E. coli as atranscription modulator to regulate transcription from lacoperator-bearing mammalian cell promoters [Brown, M. et al., Cell,49:603-612 (1987)], and those using the tetracycline repressor (tetR)[Gossen, M., and Bujard H., Proc. Natl. Acad. Sci. USA 89:5-547-5551(1992); Yao, F. et al., Human Gene Therapy, 9:1939-1950 (1998);Shockelt. P., et al., Proc. Natl. Acad Sci. USA, 92:6522-6526 (1995)].Other systems include FK506 dimer, VP16 or p65 using castradiol, RU486,diphenol murislerone or rapamycin [see Miller and Whelan, supra at FIG.2] such as FK506/rapamycin, RU486/mifepristone. Another system is theecdysone inducible system. Inducible systems are available fromInvitrogen, Clontech and Ariad. Systems using a repressor with theoperon are preferred. For example, the Idc repressor from Escherichiacoli can function as a transcriptional modulator to regulatetranscription from lac operator-bearing mammalian cell promoters [M.Brown et al., Cell, 49:603-612 (1987)] M. Gossen et al. [Proc. Natl.Acad. Sci. USA, 89:5547-5551 (1992)] combined the tetracycline repressor(tetR) with the transcription activator (VP16) to create atetR-mammalian cell transcriptional activator fusion protein, tTa(tetR-VP16), with the tetO-bearing minimal promoter derived from thehuman cytomegalovirus (hCMV) major immediate-early promoter to create atetR-tet operator system to control gene expression in mammalian cells.Recently Yao and colleagues (F. Yao et al., Human Gene Therapy, supra;Ohkawa, J., Human Gene Therapy 11 :577-585 (2000)] have demonstratedthat the tetracycline repressor (tetR) alone, rather than thetetR-mammalian cell transcription factor fusion derivatives can functionas potent trans-modulator to regulate gene expression in mammalian cellswhen the tetracycline operator is properly positioned downstream of theTATA element of a promoter such as the CMVIE promoter. One particularadvantage of this tetracycline inducible switch is that it does notrequire the use of a tetracycline repressor-mammalian celltransactivator or repressor fusion protein, which in some instances canbe toxic to cells [M. Gossen et al., Proc. Nail. Acad. Sci. USA89:5547-5551 (1992); P. Shockett et al., Proc. Natl. Acad. Sci. USA92:6522-6526 (1995)], to achieve its regulatable effects. Preferably,the repressor is linked to the target molecule by an IRES sequence.Preferably, the inducible system is a tetR system. More preferably thesystem has the tetracycline operator downstream of a promoter's TATAelement such as with the CMVIE promoter. See FIG. 4.

The effectiveness of some inducible promoters increases over time. Insuch cases one can enhance the effectiveness of such systems byinserting multiple repressors in tandem, e.g. TetR linked to a TetR byan IRES. Alternatively, one can wait at least 3 days before screeningfor the desired function. While some silencing may occur, given thelarge number of cells being used, preferably at least 1×10⁴, morepreferably at least 1×10⁵, still more preferably at least 1×10⁶, andeven more preferably at least 1×10⁷, the effect of silencing is minimal.One can enhance expression of desired proteins by known means to enhancethe effectiveness of this system. For example, using the WoodchuckHepatitis Virus Posttranscriptional Regulatory Element (WPRE). See Loeb,V. E., et al., Human Gene Therapy 10:2295-2305 (1999); Zufferey, R., etal., J. of Virol. 73:2886-2892 (1999); Donello, J. E., et al., J. ofVirol. 72:5085-5092 (1998).

A wide range of nucleic and sequences can be delivered by the presentsystem. One preferred a group of sequences encode antibodies. Antibodieshave long been used in biomedical science as in vitro tools for theidentification, purification and functional manipulation of targetantigens. Antibodies have been exploited in vivo for diagnostic andtherapeutic applications as well. Recent advances in antibodyengineering have now allowed the gene encoding antibodies to bemanipulated so that the antigen binding domain can be expressedintracelluarly. The specific and high-affinity binding properties ofantibodies, combined,” with the ability to create of large humanimmunoglobulin libraries and their ability to be stably expressed inprecise intracellular locations inside mammalian cells, has provided apowerful new family of molecules for gene therapy applications. Theseintracellular antibodies are termed “intrabodies” [W. Marasco et al.,Gene Therapy. 4:11-15 (1997)]. Preferably, the genes encode a singlechain antibody. The molecules preferably contain a tag such as HA so themolecule can be identified later.

The antibodies are preferably obtained from a library of antibodies suchas a phage display library. FIG. 6 shows a simple method to obtain theantibody and insert it into the packaging vector.

Thereafter the lentiviral vectors are used to transduce a host cell. Onecan rapidly select the transduced cells by screening for the marker.Thereafter, one can take the transduced cells and grow them under theappropriate conditions or insert those cells e.g. spleen cells or germcells, into a host animal.

The inducible promoter is turned on and one screens for cells and/oranimals displaying a particular phenotype. For example, enhancedexpression or lack of expression of a particular receptor, selectivekilling of abnormal cells, etc. The cells displaying the desiredphenotype are selected for and depending upon the phenotype, theselection can be by a high throughput automated screening. For example,beads to select cells displaying a particular receptor. FACS analysiscan be used to identify the change in expression of particularreceptors. Other systems can readily be identified. Using the tagcontained on the molecule, e.g. antibody, one can obtain the molecules.e.g., antibody that resulted in the desired phenotype. In one example,the antibody can then be used to identify the antigen it bound to, ifthat is desired.

This method permits one to use a multitude of molecules to identify aspecific molecule providing the desired function from a large group ofmolecules without first needing to know the specific identity of anymember.

In one embodiment, the vector construct uses an env gene such as VSV-G,in a VSV-G pseudotyped HIV-1 vector system in which the target moleculescomprise a very large (1×10¹⁰ member) human ER-directed sFv intrabodylibrary cloned and expressed under the control of an inducible promotersuch as the tetracycline inducible promoter system of Yao, supra. Otherenv can be used depending upon the host cell chosen. Intrabodies thatare targeted, for example, to the lumen of the ER provide a simple andeffective mechanism for inhibiting the transport of plasma membrane orsecreted proteins to the cell surface; even highly abundant cell-surfacereceptors have been reduced to undetectable levels using this method.This vector system can be used to identify sFv intrabodies that cancause “phenotypic” knockout resulting in a desired function. Forexample, killing a malignant cell, but not a corresponding normal cell,elimination of a preselected cell surface molecule, modification ofpathobiological process(s), etc. [W. Marasco et al., Gene Therapy,4:11-15 (1997)]. Moreover, since the target molecule, e.g. the sFvintrabodies, are tagged, HA-tagged, discovery and identification of thegene products knocked-out by the sFv intrabody can be readilyaccomplished through standard laboratory procedures.

Intrabodies that are intended for localization in the ER are preferablyequipped with a leader peptide and a C-terminus ER retention signal (theKDEL amino acid motif-Lys-Asp-Glu-Leu) [J. Richardson et al., GeneTherapy, 6:635-644 (1998); J. Richardson et al., Virology, 237:209-216(1997)], although other constructs can readily be made. An intermediatecloning vector allows the sFv library to be cloned directly as sFvcassettes (via for example, identical SfiI/NotI restriction sites) intoa vector which contains an immunoglobulin leader sequence, an in framecloning site for the sFvs, followed by a HA tag sequence and the ERretention sequence SEKDEL (FIG. 4). Electroporation competent TG1 cellscan be used to clone the sFv gene cassettes and obtain circa 1×10¹⁰transformants in this intermediate vector. From these transformants, theappropriate fragments, such as BamHI/XbaI fragments, will be isolated,which contain the inducible cassette, e.g., CMVtetO promoter, theER-directed sFv intrabody library, IRES and tetR. Again electroporationcompetent TG I cells are used to clone the BamHI/XbaI fragments into alentiviral vector such as an inducible system as represented byHIVNtetOIR (FIG. 5) to obtain circa 1×10¹⁰ transformants.

HIV-1 vectors expressing for example the ER-directed intrabody libraryare produced by co-transfection of for instance vectors, pCMVgag-pol,pRev and pVSV-G cDNAs into 293T cells using Superfect (Olagen) [J.Richardson et al., Gene Therapy, 6:635-644 (1998)]. Cell culturesupernatants are harvested 48-72 hours later. Ultracentrifugation areused to increase the titer of the VSV-G pseudotyped vectors and resultin obtaining titers of 10⁶ to 10⁸ infectious particles per ml. Thevectors are normalized for reverse transcriptase activity. Transductionefficiencies can be measured on CD4+ SupT cells and 293T cells by FACSanalysis of NGFR surface expression 48 hours after transduction. Forinstance, 293T cells are preferred over COS cells because they are moreefficient than COS in giving a higher titer of vectors.

For example, the resulting HIVNtetOIR vectors produced above contain alibrary of ER-directed sFv intrabodies that have the potential to cause“phenotypic” and/or “functional” knockouts because of intracellularretention/degradation of molecules that translocate through the ERincluding cell surface and secretary molecules. These vectors can beused to transduce the sFv intrabody library into CD4+ SupT cells toisolate sFv intrabodies that cause phenotypic knockout of specificmolecules that are known to be expressed on the surface of these cells.For example, CD4, CXCR4 and MHCI are expressed in high levels on thesurface of SupT cells. Other receptors can readily be chosen. Theseantibodies can also be used to target antigens that are compartments ofthe cell other than the ER-Golgi apparatus by having the leader sequencedeleted. Additionally, a target sequence such as one for the nucleus,mitochondria, etc. can readily be chosen and used in the cassettecontaining the target molecule.

Thereafter, the host cell can be transduced. For example, SupT cells areoptimally transduced and selected for the marker, e.g. NGFR expression.Preferably at least 10⁷ transduced cells are isolated by known means,e.g. beads, affinity chromatography, etc. Cells are treated with theinducer, e.g., 1 μg/ml tetracycline, and allowed to go through two tofour additional doublings so that more than one copy of each sFvintrabody gene is present in the pool of stably transduced cells.Approximately 5×10⁷ to 1×10⁸ cell in one to two ml are stained foridentification of the desired phenotype, such as with the appropriateanti-CD4, CXCR4 or MHCI Mab followed by FITC-labeled antimouse IgG. Thecells are sorted on for example, a MoFlo flow cytometer, which has highthroughput capacity (>5×10⁷ cells/ml/hr). The lowest 10% of FITC labeledcells which will include dead cells, poorly stained cells and phenotypicknockout cells are collected and expanded in tissue culture. Thisprocedure is repeated until populations of cells are recovered which areat least 50% negative for surface expression of the appropriate surfacemolecule.

Cell surface negative cells from the 50% negative pools mentioned aboveare subcloned by limiting dilution and used for further biochemical andgenetic analysis. Radioimmunoprecipitation experiments with anti-HA Mabare used to co-immunoprecipitate the target molecule. Pulse-chaseanalysis can be used to determine the half-life of the sFv/targetprotein complex. Immunofluorescence can be used to determine if thesubcellular location of the target molecule has been altered.

The target molecule, such as the sFv genes can be readily recovered byPCR or RT-PCR amplification using primers that are located in forexample the IgG leader and SEKDEL regions. The target molecule can beused to identify the ultimate target, i.e., the protein the antibodybinds to. These sFv genes are cloned into for example the pSYN bacterialexpression plasmid that contains the pelB leader sequence to direct thesFv into the periplasm, SfiI/NotI restriction sites for direct cloningof the sFv, followed by a c-myc tag and His₆ sequence. Typical yields ofsFvs recovered from periplasmic fractions of TG1 strain of E. coli thatare subsequently purified on IMAC columns range between 160 μg to 2 mgper liter from shaker flasks. These sFvs can then be used for directstaining of sFv binding of the cell surface target molecule of interest(using Mab against c-myc) or for Western blot analysis of cell extracts.One can also use nanosequencing or GC mass spec to identify a sequenceor protein (e.g., a target) where only a small amount of the product ispresent. See. e.g., Jin, Y., et al., J. of Biol. Chem. 274:28301-28307(1999) at 28304-305.

The lentiviral virion (particle) is expressed by at least one vectorcontaining a nucleic acid sequence encoding the lentiviral pol and gagproteins necessary for viral protein expression operably linked to apromoter. Preferably, multiple vectors are used. Preferably, the polsequences encoding pol proteins are on more than one vector. There isalso a vector having nucleic acid sequence encoding the lentiviral gagproteins necessary for reverse transcription and integration operablylinked to a promoter. Preferably, this gag nucleic acid sequence is on aseparate vector than the pol nucleic acid sequence. The use of separatevectors for the various “genes” further reduces the chance of areversion to wild-type.

In one embodiment, the lentiviral vector is modified so that the gagsequence does not express a functional MA, protein, i.e. it is MA. Thiscan be accomplished by inactivating the “gene” encoding the MA byadditions, substitutions or deletions of the MA coding region. Since theMA is part of the gag gene and as expressed, is processed from theprecursor protein, when referring to a MA gene (or coding region), weare only referring to that portion of the entire gag gene that encodesthe MA subunit. Preferably, the inactivation is accomplished bydeletion. Preferably, at least 25% of the MA coding region is deleted.more preferably, at least 50% is deleted, still more preferably, atleast 60%, even more preferably at least 75%, still more preferably, atleast 90%, yet more preferably at least 95% and most preferably theentire coding region is deleted.

The MA has a myristylation anchor and that myristylation anchor(sequence) is required. Preferably, the mvristylation sequence is aheterologous (i.e., non-lentiviral) sequence. Src, MARCKS(myristolylated alanine-rich C kinase substrate), ARF (ADP-ribosylationfactor), recovering and related EF-hand calcium-binding proteins(visinin neurocalcium and others), and non-lentiviral gag proteins(e.g., Moloney murine leukeemia virus. Mason-Pfizer monkey virus).

The MA-deleted viruses consistently exhibit an increased ability torelease extracellular virus particles, indicating that there is norequirement for the globular domain of MA for stable membraneassociation. Surprisingly, deleting the globular head of MA, whichharbors the putative MA nuclear localization signal (NLS), also permitsthe early steps of the lentiviruses replication cycle in macrophages.

In one embodiment the env nucleic acid sequence encodes a lentiviralenvelope protein. When using a MA- gag vector, it is preferred that theenv sequence is altered from the wild type sequence so that it encodes atruncated cytoplasmic tail. Preferably, 50% of the cytoplasmic tail ismissing. More preferably, at least 75% is deleted still more preferablyat least 90% is deleted, even more preferably, at least 95% is deleted.Most preferably, the entire cytoplasmic tail is deleted in such anembodiment.

In another embodiment the lentiviral vector is another form ofself-inactivating (SIN) vector as a result of a deletion in the 3′ longterminal repeat region (LTR). Preferably, the vector contains a deletionwithin the viral promoter. The LTR of lentiviruses such as the HIV LTRcontains a viral promoter. Although this promoter is relativelyinefficient, when transactivated by e.g. tat, the promoter is efficient.However, the presence of the viral promoter can interfere withheterologous promoters operably linked to a transgene. To minimize suchinterference and better regulate the expression of transgenes, thelentiviral promoter is preferably deleted.

Preferably, the vector contains a deletion within the viral promoter.The viral promoter is in the U3 region of the 3′ LTR. A preferreddeletion is one that is 120 base pairs between Scal and Pvul sites, e.g.corresponding to nucleotides 9398-9518 of HIV-1 HXBC encompassing theessential core elements of the HIV-1 LTR promoter (TATA box, SP1 andNF—Pb binding sites). The further 5′ you go downstream the more dramaticthe “SIN” effect. Indeed, deletions of up to 400 base pairs have proveneffective. Zuffrerey, R., et al., J. of Virol 72:9873-9880 (1998). Afterreverse transcription, the deletion is transferred to the 5′ LTR,yielding a vector/provirus that is incapable of synthesizing vectortranscripts from the 5′ LTR in the next round of replication. Thus, thevector of the present invention contains no mechanism by which the viruscan replicate as it cannot express the viral proteins.

In another embodiment the vector is a tat deleted vector. This can beaccomplished by inactivating at least the first exon of tat by knowntechniques such as deleting it or introducing a stop codon.Alternatively, one can extend the U3 LTR deletion into the R region toremove the TAR element. The tat deleted vectors result in high titer ofvirus.

Variations can be made where the lentiviral vector has multiplemodifications as compared to a wildtype lentivirus. For example, withHIV being nef-, rev-, vif- and vpr-. In addition one can have MA-gag, 3′and 5′U3 deleted LTR and variations thereof.

By using these techniques a number of safety reduncancies are introducedinto the vectors.

In a more preferred embodiment, the env sequence encodes an envelopeprotein from a different virus than the lentiviral gag and pol genes.The resultant particle is referred to as a pseudotyped particle. Byappropriate selection of the envelope protein one can transformvirtually any cell. Preferably envelopes are influenza virus, VSV,filoviruses such as Ebola, more preferably VSV-G or Ebola.

As discussed above, the envelope protein chosen can effect the hostrange. For example, a VSV-G pseudotyped particle will infect a widerange of cells. Whereas Ebola pseudotyped particles or HTLV-pseudotypedparticles have a limited host range.

While env glycoproteins are dispensable for particle production per se,their ion is required for the formation of infectious virions.

The vector system can be used to package a wide range of desirednucleotide preferably a RNA segment, into an empty lentiviral particlebecause of the large of lentiviruses. In addition, the use of promotersand enhancers can also significantly length of an insert. Preferably,the system is used with groups containing multiple molecules displayingdiversity such as genetic diversity. Accordingly, the system of thepresent invention provides a significant advantage over currentlyavailable vectors by allowing for inserts that can contain a number ofpromoters and genes and that can be used to transfect resting cells aswell as dividing cells.

The vector(s) is prepared so that none of the nucleotide segments usedwill contain a functional packaging site containing sequence. (Thissequence is referred to as the packaging sequence.)

The vector(s) do not contain nucleotides from the lentiviral genome thatpackage lentiviral RNA, referred to as the lentiviral packagingsequence. In HIV this region corresponds to the region between the 5′major splice donor and the gag gene initiation codon (e.g., nucleotides301-319 in HXB2). Preferably, these vectors also do not have lentiviralLTRs such as the HIV LTR. The env, gag, and pol genes are operablylinked to a heterologous promoter.

The packaging sequence can be excluded from the vector(s) by any of avariety of techniques well known to the person of ordinary skill in theart. For example, one can simply delete the entire sequence.Alternatively, one can delete a sufficient portion of a sequence torender it incapable of packaging. An alternative strategy is to insertnucleotides into such a site to render it non-functional. Mostpreferably, one will delete the site entirely to prevent homologousrecombination.

Accordingly, the lentiviral vectors can express the desired viralproteins, but because the packaging site has been removed and thelentiviral LTRs are not operational, their mRNA will not be effectivelypackaged into the lentiviral particles, and the recombinant virus willnot be able to replicate and infect other cells.

The lentiviral vectors can also contain sequences encoding desiredlentiviral regulatory proteins such as Tat, Rev, etc. However, in anumber of embodiments it is preferable not to contain such regulatorygenes. If RRE and CAR sequences are included in the gene, the inclusionof sequence encoding RREV is necessary in this, unless the virus isexpressed in the cytoplasm. Alternatively, you can use constitutivetransport elements (CTE) in place of RRE to make the vectors REVindependent. Also, there is less sequence homology. Srinivasakumar, S.,et al., J. of Virol., 73:9589-9598 (1999); Srinivasakumar, et al., J. ofVirol., 71:5841-5848 (1997). These regulatory sequences can be on theother lentiviral vectors (e.g., gag vector, pol vector, gag-pol vectoror env vector), or on their own lentiviral vector.

A desired heterologous nucleic acid segment can be encapsulated withinthe empty lentiviral particle by using a vector containing a nucleicacid segment of interest and a lentiviral packaging sequence necessaryto package lentiviral RNA into the lentiviral particles at the time thelentiviral vectors are used. Preferably, the vector contains a 5′ and 3′lentiviral LTR with the desired nucleic acid segment inserted betweenthem. The nucleic acid segment preferably encodes a protein.

An origin of DNA replication (ori) which is recognized by the viralreplication proteins and enzymes may also be present. This vectorpermits packaging of desired nucleotide inserts in the pseudotypedparticles. This vector is sometimes referred to as the packaging vector.This packaging vector is used to package any group of desiredheterologous nucleic acid sequence, preferably a RNA sequence, into theparticle. Preferably, the packaging vector contains (a) a promotersequence operably linked to at least one heterologous nucleic acidsequence and (b) at least one sequence sufficient to permittranscription and processing of mRNA, the translation of which resultsin an expressed protein. Preferably, the processing sequence is apolyadenylation sequence. Preferably the promoter is part of aninducible system. Still more preferably, this vector contains anintervening sequence following the promoter sequence. Preferably thesequences containing the promoter, target molecule, and optionally arepressor sequence also contains a tag such as HA to permit readyidentification of the target molecule. This grouping of elements issometimes also referred to as the cassette. For example, theheterologous sequence can encode any desired protein, preferably atherapeutic protein or an antibody. It can also encode antisense DNA,RNA or a desired immunogen, such as an antigenic protein. It can encodespecific peptide sequence that will generate an immunogenic reaction.Such a peptide sequence is typically at least about 6 amino acids inlength.

The heterologous nucleotide sequence can encode a wide variety ofproteins such as a therapeutic protein, i.e., one that compensates foran inherited or acquired deficiency. Examples of therapeutic proteinsinclude neurotransmitter biosynthetic enzymes, e.g., tyrosinehydroxylase for the treatment of Parkinson's disease; neurotrophicfactors including neutrophins, e.g., nerve growth factor for thetreatment of Alzheimer's disease, one can also use nerve growth factorreceptor and the trk receptor; hypoxanthine-guanine porphoribosyltransferase (HGPRT) for the treatment of Lesch Nyhan disease;β-hexosaminadase a chain for the treatment of Tay Sachs disease; insulinfor the treatment of diabetes; angiogenic proteins such as VEGF, bFGFfor the treatment of ischemic tissues, circulatory problems, etc.,antianglogenic proteins for treating malignant cells such as plateletfactor IV, endostaten, etc. Receptors can also be delivered, e.g. thenerve growth factor receptor, the trk receptor, etc. Because the insertcan be large, it is possible to encode a series of different proteins.For example, one can encode a series of proteins that form areceptor-ligand complex.

Other proteins include, for example, signal transduction enzymes, e.g.,protein kinase c; transcription factors, e.g., c-fos, NF-PB; oncogenes,e.g., erbB, erbB-2/neu, ras; neurotransmitter receptors, e.g., glutamatereceptor, doparmine receptor, etc.

One preferred group of proteins that are encoded are angiogenicproteins. As used herein the term “angiogenic protein” means anyprotein, polypeptide, mutein or portion that is capable of, directly orindirectly, inducing blood vessel growth. Such proteins include, forexample, acidic and basic fibroblast growth factors (aFGF and bFGF),vascular endothelial growth factor (VEGF) including VEGF-1 and2,-epidermal growth factor (EGF), transforming growth factor I and θ(TGF-I and TGF-θ), platelet-derived endothelial growth factor (PD-ECGF),platelet-derived growth factor (PDGF), tumor necrosis factor I (TNF-I),hepatocyte growth factor (HFG), insulin like growth factor (IGF),eryhropoietin, colony stimulating factor (CSF), macrophage-CSF (M-CSF),granulocyte/macrophage CSF (GM-CSF), angiopoetin-1 (Ang1) and nitricoxidesynthase (NOS). See, Klagsbrun, et al., Annu. Rev. Physiol.,53:217-239 (1991); Folkman, et al., J. Biol. Chem., 267:10931-10934(1992) and Symes, et al., Current Opinion in Lipidology, 5:305-312(1994). Muteins or fragments of a mitogen may be used as long as theyinduce or promote blood vessel growth.

VEGF is a preferred angiogenic protein. VEGF-1 exists in severaldifferent isoforms that are produced by alternative splicing from asingle gene containing eight exons (Tischer, et al., J. Biol. Chem.,806, 11947-11954 (1991), Ferrara, Trends Cardio. Med., 3, 224-250(1993), Polterak, et al., J. Biol. Chem., 272, 7151-7158 (1997)). HumanVEGF isoforms consists of monomers of 121 (U.S. Pat. No. 5,219,739),145, 165 (U.S. Pat. No. 5,332,671), 189 (U.S. Pat. No. 5,240,848) and206 amino acids, each capable of making an active homodimer (Houck, etal., Mol. Endocrinol., 8, 1806-1814 (1991)). Other forms of VEGF includeVEGF-B and VEGF-C (Joukou, et al., J. of Cell. Phys. 173:211-215 (1997),VEGF-2 (WO 96/39515), and VEGF-3 (WO 96/39421).

Another preferred group of encoded proteins are antibodies. Included aredAbs, single chain antibodies, Fabs. Single chain antibodies arepreferred. Libraries of antibodies are known and can be used in thepresent invention. For example, using a phage display library bothgeneralized and specialized libraries can be used. A specialized librarywould be one where the member antibodies are generated to a specificgroup of antigens, e.g. a specific tumor. The diversity of the membersof a specialized library is less than that of a generalized library.

The heterologous nucleotide sequence can also encode antisense molecules(DNA or RNA). These molecules can be used to regulate gene expressionassociated with a particular disease. The antisense molecules areobtained from a nucleotide sequence by reversing the orientation of thecoding region with regard to the promoter. Thus, the antisense RNA iscomplementary to the corresponding mRNA. For review of antisense sciencesee Green, et al., Ann. Rev. Biochem. 55: 569-597 (1986), which isherein incorporated by reference. The antisense sequence can containmodified sugar phosphate backbones to increase stability and make themless sensitive to RNA sensitivity. Examples of the modifications aredescribed by Rossi, et al., Pharmacol. Ther. 50(2):245-354 (1991).Another class of molecule includes ribozymes. Ribozymes and antisensemolecules that engage in, as well as those that do not show transplicingcan be used.

The heterologous nucleotide sequence is preferably operably linked to apromoter sequence capable of directing transcription of the sequence ina desired target cell. Lentiviruses such as the primate lentivirusescontain the Tat regulatory protein. This protein will transactivate aprotein operably linked to a TAR element. The TAR element is present inthe 5′ LTR of the primate lentivirus. Thus, the expression ofheterologous protein can be enhanced by transactivation. The LTR alsocontains a promoter. However, that promoter in the absence oftransactivation is relatively ineffective. Thus, the use of otherpromoters and enhancers is typically preferred. The promoter can be apromoter such as the SV40, CMV, HSV-1 IE, IE 4/5 or RSV (Rous sarcomavirus) promoters. Others include Srα-promoter (a very strong hybridpromoter composed of the SV40 early promoter fused to the R/U5 sequencesfrom the HTLV-I LTR), tetracycline-regulatable promoters,tissue-specific promoters (e.g., alpha-fetoprotein promoter; andrhodopsin promoter for photoreceptor-targeted expression). Otherpromoters capable of directing transcription of the heterologoussequence in a specific target cell can also be used to more specificallydirect expression of the heteroloous gene to a desired target (host)cell. Indeed, one can link the inducible promoter construct with atissue specific promoter. For example, if the target cell is a neuronalcell, a promoter such as the neuron specific enolase promoter[Forss-Petter. et al., J. Neurosci. Res. 16: 141-56 (1986)] can be used.The rat tyrosine hydroxylase (TH) promoter can support cell typespecific expression in the midbrain [S. Song et al., J. Neurochem: 68:1792-803 (1997)]. Furthermore. the use of inducible promoters or otherinducible regulatory sequences, which are well known in the art, in someembodiments are preferred. For example, the tetR-tetO system. Asdiscussed the promoter in the LTR can interfere with the other promoter.Thus, in certain embodiments it is preferable to inactivate the viralLTR promoter.

In order to minimize the possibility of a recombination event betweenthe packaging vector that transfers the desired heterologous gene(s) andthe lentiviral vector generating a wild type lentivirus, it is desirablethat the packaging vector has a minimal degree of homology with thenucleotide segments encoding the particle vector. Preferably, one woulduse different promoters in these different vectors. These goals can beaccomplished by a variety of means known in the art based upon thepresent disclosure. For example, in order to minimize any chance ofrecombination, it is preferable to use multiple vectors. Additionally,it is preferable to reduce the chance of homologous recombination byminimizing sequence overlap. For example, one can delete unnecessarylentiviral sequences. Alternatively or additionally, one can use knowntechniques to change the nucleotide sequence of the vectors. One methodof accomplishing this is referred to as nucleotide, e.g., DNA,shuffling. One changes nucleotides in codons, e.g., the third base ofeach codon within the lentiviral constructs of one vector. Thus, thesame coding sequence in a second vector now differs and will not besubject to homologous recombination. Changes in the codons of thevarious vectors can be made to optimize nucleotide differences.

Alternatively or in combination with the above approach of reducinghomology, one can alter the sequence of a gene from the lentivirussegment so that it does not encode a functional protein. As used herein“functional” means a protein having wild-type activity.

Depending upon the particular purpose for the particles one can useknown techniques to alter the lentivirus segment to inactivate genesthat encode proteins present in the particle which cause certaineffects. For example, inactivating those proteins that enhancereplication, e.g., rev and/or tat. Vpu affects infectivity. Nef alsoaffects the virus. It has been reported that nef appears to be requiredfor efficient replication in vivo.

Cells can be transfected by the vectors to prepare the viral particle.One can prepare the vectors in vitro, one would then harvest theparticles, purify them and inject them by means well known in the art.More preferably one would purify the particles, and then use those toinfect the desired cells.

One can create producer cell lines expressing virions and transform suchcells with the packaging vector. The producer cell lines or any cell canbe transformed by standard techniques. One preferred method is to use aninactivated adenovirus vector linked to the packaging vector by acondensing polycation such as polylysine or polyethylanimine (PEI) [seeBaker, A. et al., Nucleic Acids Res., 25(10):1950-1956 (1997); Baker, A.et al., Gene Ther., 4(8):773-782 (1997); Scaria, A. et al., Gene Ther.,2:295-298 (1995)]. The use of PEI is a condensing polycation ispreferred.

As used herein, the packaging vector refers to the vector that containsthe heterologous gene to be transferred under the control of a promoter(e.g., internal tissue-specific or inducible) flanked by lentiviralLTRs, and the packaging and leader sequences necessary for encapsidation(i.e., packaging). This vector is sometimes referred to in theliterature as transfer vector and it is the constructs encoding theproteins and enzymes required for encapsidation that are referred to asthe packaging construct.

The vectors express proteins and mRNA which assemble into particles andhence can be used to express large amounts of viral particles. Thisrequires transfecting a cell with the particle vector system describedherein, the packaging vector, and culturing the cell line underconditions and time sufficient to express the viral proteins which thenform the particles. Thereafter, the particles can be purified by knowntechniques with care taken to insure that the structure of the particleis not destroyed. The particles can be used in a variety of areas. Forexample, they can be used to generate a desired immune reaction totransform a cell with a heterologous nucleic acid sequence and/or todeliver a nucleic acid sequence to a desired host cell.

One can prepare transient or stable cell lines that express thelentiviral particles by standard techniques based upon the presentteaching.

Thereafter, if stable cell lines are desired, one can screen for thosecells that have been stably transfected by standard technique.

Such stable producer cell lines are a preferred source for obtainingpackaged particles.

The particles of the present invention can be used to deliverheterologous DNA to a target cell. The target cell may be in vivo, invitro or ex vivo. The target cell can be a dividing or preferably aquiescent cell. Quiescent cells include nonmitotic or postmitotic cells.The preferred nonmitotic cell is a macrophage. The target cells alsoinclude cells of the nervous system, e.g., neural or neuronal cells.Preferred quiescent or slowly dividing target cells include glia cells,myocytes, hepatocytes, pneumocytes, retinal cells, and hematopoieticstem cells. Pancreatic islet cell are also a preferred target.

In the present method the use of in vitro cells in presently preferred.However, there are instances where in vivo or ex vivo administration isdesirable.

Introduction of the viral particle carrying the heterologous gene to bedelivered to a target cell may be effected by any method known to thoseof skill in the art. For example, with in vivo administration, thefollowing techniques are preferred. Catheters, injection, scarification,etc. For example, stereotaxic injection can be used to direct the viralparticles to a desired location in the brain. Stereotaxic surgery isperformed using standard neurosurgical procedures [Pellegrino and Clapp,Physiol. Behav. 7: 863-8 (1971)]. Additionally, the particles can bedelivered by intracerebroventricular (“icv”) infusion using a minipumpinfusion system, such as a SynchroMed Infusion System. A recent methodbased on bulk flow, termed convection, has also proven effective atdelivering large molecules to extended areas of the brain and may beuseful in delivering the viral particle to the target cell [R. Bobo etal., Proc. Natl. Acad Sci. USA 91: 2076-80 (1994); P. Morrison et al.,Am. J. Physiol. 266: R292-305 (1994)]. Other methods can be usedincluding catheters, intravenous, parenteral, intraperitoneal andsubcutaneous injection, oral or other known routes of administration.

In some instances one would use these vectors to transform host cells invivo to look for effects of specific genes in a living system. One wouldinject a sufficient amount of the separate vectors or preferably thepackaged viral particles to obtain a serum concentration in the tissuecontaining the target cell of the therapeutic protein ranging betweenabout 1 pg/ml to 20 μg/ml. More preferably between about 0.1 μg/ml to 10μg/ml. Still more preferably, between about 0.5 μg/ml to 10 μg/ml. Forexample, by expressing a specific protein or alternatively, by blockingthe function of a protein such as by expressing an antibody to aspecific sequence intracellularly.

For example, solid dose forms that can be used for oral administrationinclude capsules, tablets, pills, powders and granules. In such soliddose forms, the active ingredient, i.e., empty virus particle, is mixedwith at least one inert carrier such as sucrose, lactose or starch. Suchdose forms can also comprise additional substances other than inertdiluents. e.g., lubricating agents, such as magnesium stearate.Furthermore, the dose forms in the case of capsules, tablets and pillsmay also comprise buffering agents. The tablets, capsules and pills canalso contain time-release coatings to release the particles over apredetermined time period.

For parenteral administration, one typically includes sterile aqueous ornonaqueous solutions, suspensions or emulsions in association with apharmaceutically acceptable parenteral vehicle. Examples of non-aqueoussolvents or vehicles are propylene glycol, polyethylene glycol,vegetable oils such as olive oil and corn oil, gelatin and injectableorganic esters, such as ethyl oleate. These dose forms may also containadjuvants such as preserving, wetting, emulsifying and dispersingagents. They may be sterilized by, for example, filtration through abacterial-retaining filter, by incorporating sterilizing agents into thecomposition, by irradiating the compositions, etc, so long as care istaken not to inactivate the virus particle. They can also bemanufactured in a medium of sterile water or some other sterileinjectable medium before use. Further examples of these vehicles includesaline, Ringer's solution, dextrose solution and 5% human serum albumin.Liposomes may also be used as carriers. Additives, such as substancesthat enhance isotonicity and chemical stability, e.g., buffers andpreservatives, may also be used.

The preferred range of active ingredient in such vehicles is inconcentrations of about 1 mg/ml to about 10 mg/mil. More preferably,about 3 mg/ml to about 10 mg/ml.

EXAMPLES

Construction of a Single-Plasmid Tetracycline-Inducible System

Basic One-Plasmid System

Our single inducible cassette (outlined in FIG. 1A) was constructed bythree piece ligation of a internal ribosomal entry site (IRES) from theencephalomyocarditis virus (EMCV) and the tetR fragment removed frompcDNA3tetR into a NotI/ClaI sites of pCMVtetOhEGF [F. Yao et al., HumanGene Ther. 9: 1939-1950 (1998)]. The plasmid pCMVtetOhEGF, used as theparental vector for all our constructs contains the human epidermalgrowth factor (hEGF) gene driven by a chimeric promoter composed of ˜650bp of the immediate early enhancer, cytomegalovirus promoter (ieCMV) andtwo tandem repeats of the tetracycline operator (tetO) positioned 10 bpdownstream of the TATA box. A NotI-NheI fragment encoding the IRESsequence was removed from a previously described vector, pCMV-Fab105/21H previously prepared by R. Levin et al., Mol. Med. 3: 96-110(1997). A subcloning step, using the intermediate pGem7Zf(+) vector(Promega, Madison, Wis.), was required to clone the XbaI-EcoRItetR-containing fragment from pcDNA3tetR allowing the introduction ofthe flanking restriction sites (NheI-ClaI) necessary for the finalcloning step as well as the insertion of a Kozak sequence preceding thefirst ATG [M. Kozak, J. Mol. Biol. 196: 947-950 (1987)]. Previous tothis step, pGEM7Zf(+) vector was modified by incorporating a syntheticlinker containing aHindIII-NheI-Kozak(CCACC)-ATG-XbaI-EcoRI-Stop(TATTAA)-SpeI-ClaI-SphIrecognition sites. A pair of oligonucleotides carrying the correspondingsequence was synthesized and equivalent amounts of each (10 μg) werehybridized prior to the final ligation into the HindIII-SphI sites ofpGEM7Zf(+) vector. The resulting 0.65 Kb NheI-ClaI-tetR fragment wasinserted downstream of the IRES sequence and prior to thepolyadenylation site of the pCMVtetOEGF vector. This position allowscap-independent translation of tetR from the single mRNA transcript. Thefinal three piece ligation step was performed using a DNA ligation kitfrom Takara and according to manufacturer procedures. Similarly, apCMVhEGF plasmid lacking the tetO was modified to incorporate the IRESsequence and the tetR gene for its use as non-regulatable controlplasmid.

Introduction of a Nuclear Localization Signal

A three tandem repeat sequence corresponding to the nuclear localizationsignal (NLS) from simian virus large T-antigen(GATCCAAAAAAGAAGAGAAAGGTA) was incorporated at the 3′ end of tetRpreceding the stop. A pair of complementary oligonucleotides containingthe nls sequence were synthetically prepared and, after hybridization,cloned in frame between the EcoRI and SpeI sites of pGEM7Zf(+)-tetR.Then, constructs previously described were modified by replacing thetetR gene for the tetR.NLS fragment.

Replacement of the hEGF Reporter Gene by eGFP Gene

The BamHIII/NotI fragment containing the hEGF gene was excised from thebasic inducible system and replaced by the enhanced green fluorescentprotein (eGFP) gene. The 700 bp fragment encoding eGFP was removed frompeGFP.IRES.neo vector (Clontech. Palo Alto, Calif.) and directly ligatedinto the parental constructs.

The final plasmids were purified using the Endotoxin-free Maxi Kit fromQiagen Inc. (Valencia, Calif.).

In Vitro Functionality of Our Single-Plasmid System Versus the OriginalTwo Component Inducible System.

Cell Culture and Transfections

African green monkey kidney cells, Vero. COS-1 and COS-7 cell lines andhuman kidney 293-T cells were grown and maintained in Dulbecco'smodified Eagle's medium (D-MEM) (GIBCO-BRL, Grand Island, N.Y.)supplemented with 10% fetal bovine serum (Tissue Culture Biologicals,Tulare, Calif.) and antibiotics. D-MEM media containing 10% of the TetSystem approved fetal bovine serum (Clontech, Palo Alto, Calif.) wasused for functional testing of our inducible system.

The day before transfection, cells were subcultured into six-well plates(Becton Dickinson, Franklin Lakes, N.J.) at densities of 2×10⁵cells/well. Transient transfection assays were performed using theSuperfect reagent (Qiagen, Valencia, Calif.) as described by themanufacturer. DNA complexes were prepared using 2.5 μg of plasmid DNAand Superfect reagent at a 1:2 ratio of DNA to condensing agent,followed by incubation at room temperature for 10 min and finally,addition of the complexes to the cells. Comparison with the 2 plasmidsystem was carried out using 0.5 μg of pCMVtetOhEGF or pCMVhEGF. in eachcase alone or in combination with 2 pg of pcDNA3tetR or empty vectorDNA, pcDNA3.1(−). After 18 hr incubation at 37° C. in a humidifiedatmosphere of 5% CO₂, cells were washed with PBS and refed with freshmedia in the presence or absence of tetracycline (1 μg/ml). Reportergene expression was measured as a function of time after transduction aswe detailed in another section.

Evaluation of Reporter Gene Expression

Expression of hEGF in cultured media was performed by the ELISAtechnique. Briefly, 96 well plates were coated with an anti-hEGFmonoclonal antibody (MAB236; R&D Systems, Minneapolis, Minn.) (100ng/well) at room temperature (RT) for 5 hr and then blocked using 3%non-fat milk in phosphate saline buffer (PBS). Samples extracellularmedium and recombinant HEGF standards prepared in a two-fold dilutionseries ranging from 9.7-5,000 μg/ml (234-EG; R&D Systems) were incubatedat 4° C. overnight. A secondary polyclonal antibody specific to HEGF(sc275; Santa Cruz Biotechnologies. Santa Cruz. Calif.), was then added(10 μg/well) and incubated for 2 hr at RT. The horseradish peroxidase(HRP)-conjugated goat anti-rabbit polyclonal antibody (sc2004; SantaCruz) was the tertiary antibody (3.33 ng/well). Finally, the peroxidaseassay was performed (Bio-Rad, Hercules, Calif.), according tomanufacturer's procedures and the reactions analyzed on a microplatereader (Molecular Devices, Sunnyvale, Calif.).

Production of the green fluorescent protein from constructs bearing theeGFP gene was detected by FACS analysis and histochemistry.

RNA Extraction and Northern Blot Analysis

Total cytoplasmic RNA was extracted from transfected cells using theTRIzol Reagent (GIBCO-BRL) and according manufacturer's procedures. RNA(20 μg) was separated on 1.2% formaldehyde/agarose gels and transferredto nylon Hybond-N filter membranes (Amersham, Arlington Heights, Ill.)by pressure blotting. Blots were probed with a XbaI-EcoRI tetR DNAfragment (25 ng) labeled using the Megaprime DNA labeling system(Amershan) and [³²P]-dCTP (NEN, Boston, Mass.). Overnight hybridizationwas performed using 4×10⁷ cpm of labeled probe in a solution containing0.5%[w/v] SDS. 5× Denhardt's solution [0.1% BSA, 0.1% Ficoll, 0.1% PVP]and 5×SSPE [0.9M NaCl, 50 mM sodium phosphate. 5 mM EDTA, pH7.7] at 42°C. Blots were washed at a final stringency of 0.1% SDS, 0.1% SSPE at 60°C. and then visualized by autoradiography after exposure at −80° C.

Immunolocalization of tetR in Transfected Cells

Vero cells (5×10⁴/well) were plated the day before transfection onchamber glass slides. Transfection of the constructs was performed asdescribed above. Forty-eight hours after treatment (plus or minus 1μg/ml tet), cells were fixed with 4% formaldehyde in PBS for 20 min atRT. Upon fixation, cells were permeabilized with 0.2% Triton X-100 for 5min at RT and blocked with 10% normal goat serum, 5% BSA in PBS for 30min. A monoclonal antibody raised against tetR (Clontech) was added in a1:100 dilution and incubated for 1-2 hrs. A goat anti-mouse IgG coupledto FITC (Sigma. St. Louis, Mo.) or alternatively labeled with PE(Boeheringer Mannheim) at 1:250 dilution was added to the cells andincubation continued for an hour. After washing with PBS, coverslipswere mounted in Sigma medium and examined under the UV light using afluorescence microscope (Nikon Diaphot 300) with FITC and Rhodamineexchangeable filters. Images recorded in a spot cooled color digitalcamera were analyzed using the Oncor Image software and printed fromAdobe Photoshop, V3.0 for Macin tosh.

Preparation and Evaluation of HIV-1-Based Vectors

The vectors used are based on the HIV-1 proviral clone HXB2 (FIG. 1). Amore detailed description of the basics for our viral vectorconstruction has been previously reported by Richardson et al., GeneTher., 5:635-644 (1998).

The original multiple attenuated vector (with deletions in the nef, rev,vif and vpr genes, HVPΔEB) was modified to silence transcriptionalactivation from the viral promoter region which can otherwise causeinterference of transgene expression when using internal promoters(promoter interference). The resulting self-inactivated (SIN) transfervector or HVPΔEBΔLTR was generated by a simple ScaI/partial PvuIIdigestion and insertion of a PacI linker, eliminating therefore a 120 bpfragment (nucleotides 9398-9518) encompassing the TATA box, SP1 andNF—KB sites on the 3′LTR. The sequence of the modified U3 region in thetransfer plasmid was confirmed by DNA sequencing.

A novel improved version of the original vector was generated by a 2.5Kb deletion (nucleotides 830-2096 and 5743-7041) into the remaining gagregion and the first exon of the tat and rev genes (NVPAEBAtat). Thisfragment was removed by ClaII/ClaI digestion and consequent re-ligation,resulting in a tat-vector.

To determine the transduction efficiency of the three developed vectors,the enhanced green fluorescent protein (eGFP) either under control ofthe CMV promoter or in absence of any internal promoter was introducedinto the transfer vectors. A synthetic linker containing aBamHI-MluI-NotI-XbaI-XhoI sites was inserted into the plasmid vectors toincorporate the suitable cloning sites and then the MluI-NotI CMV EGFP(Clontech, Palo Alto, Calif.) or the BamHI-NotI EGFP fragments weremoved into the vectors. Viral vector packaging and transduction

The pseudotyped HIV-vector particles were produced in COS-1 cells(˜1.5×10⁶ cells/10 mm dish) by transient co-transfection of the transfervector (5 μg), packaging plasmid (2.5 μg). VSV-G-(1 μg) andrev-expressor (1 μg) plasmids using Superfect reagent (Qiagen) accordingto manufacturer's instructions. Medium was replaced after 24 hours andvirus was harvested 36-48 hours later. The conditioned media wasscreened for reverse transcriptase activity and 1 ml was used totransduce 1×10⁶ Hela cells. Transduction efficiency was determined byfluorescent-activated cell sorting (FACS) analysis.

Construction of an Inducible HIV-1 -Based Vector

Replication-deficient VSV-G pseudotyped HIV-1 vectors were generated bytransient cotransfection of 293T human kidney cells using three plasmidcombination. They consist of a helper construct encoding for theproteins and enzymes necessary for lentiviral production, anenvelope-expressor and the transfer vector.

The transfer vector plasmid is devoid of most of the gag-pol andenvelope genes but maintains the cis-acting elements necessary forencapsidation, reverse transcription and integration. The pHlibCMVeGFP(wild-type) vector contains a 3.1 Kb deletion into the gag-pol regionand two deletions into the env gene region (1.5 and 0.55 Kb) that allowsinsertion of a foreign gene as well as makes the virus non-replicative.To study the ability of the lentiviral vectors to infect and provideefficient gene expression we have used the enhanced green fluorescentprotein (eGFP) gene as a marker gene. hEGF or any other marker could beused instead. Vectors containing the eGFP gene under the control ofeither the heterologous immediate early CMV promoter or the viral 5′ LTRwere prepared by standard techniques.

Improvement in vector biosafety was achieved by constructing aself-inactivated vector (SIN vector) by introducing a 120bp deletion inthe 3′LTR region (9398-9518 bp) of the wild type vector. During reversetranscription, the missing DNA fragment is transferred to the 5′ LTRregion resulting in a deletion of the TATA box. SP1 and NF-kB cis-actingelements that will consequently lead to viral promoter attenuation inthe resulting proviral DNA.

We also generated a tat-independent vector by site directed mutagenesis.A three base mutation within the first two codons of the first exon ofthe tat gene was introduced. resulting in a two amino acid substitution(the first aa, Met to Ile and the second. Glu to a Stop signal).

Two other plasmids required to build an HIV-1 based vector are thepackaging construct. pCMV R8.2, and an envelope-expressor plasmid. Thesetwo plasmids don't contain any of the HIV-1 packaging elements(packaging signal and LTR) necessary for encapsidation and/orintegration. Expression of helper's proteins is under regulation of theimmediate early CMV promoter and transcription termination is providedby the SV40 polyadenylation signal. For different applications weprepared pseudotyped viruses containing the vesicular stomatitis virusglycoprotein (VSV-G), or alternatively, the Ebola glycoprotein (Eb-GP).Recombinant virus generated by three plasmid co-transfection contain theelements required for reverse transcription, integration and geneexpression but won't be able to support replication.

Transient cotransfection of 293T cells was carried out by theconventional calcium phosphate technique. Supernatants harvested after48-60 hr incubation were cleared by passing the cultured media through a0.45 or 0.22 μm filter and then, virus was concentrated byultracentrifugation at 100,00×g for 45 min. An alternative concentrationprocedure involved the use of a 100,000 MW cut-off filter during aconventional centrifugation step. Reverse transcriptase (RT) levels weretested in aliquots harvested before and after concentration as aparameter for viral concentration and in parallel. eGFP expression on293T cells or in HeLa cells determine to establish the actualtransducing units in the final preparation.

Construction of an Inducible HIV-1 Vector

Our single tetracycline-inducible and control bicistronic cassettes wereremoved from the eukaryotic cloning vector with the appropriaterestriction sites and cloned into the SIN vector carrying the tatmutation. In this way, any interference of Tat protein over the internalCMV promoter was avoided.

Construction of a Very Large, Naive, Human sFv Phage Display Library

A large, naive, human sFv library was constructed by performing 80electroporations of >275 million human V_(H)genes randomly combined with1.6 million each of V_(kappa)- and V_(lambda)-gene III fusions in thepFARBER phagemid vector. These ratios were chosen to maintain maximalV_(H)diversity since the majority of binding energy is contributed byV_(H)CDR3. A total of 1.63×10¹⁰ transformants were isolated. Analysis byrestriction enzyme digestion demonstrated an sFv insert efficiencyof >92%, yielding a library of 15 billion members. This library wasreadily rescued with helper phage and infected TG 1 bacteria allcontained the expected 800 bp sFv insert. Master vials of thetransformed bacterial were aliquoted and frozen as glycerol stocks.

Analysis of Genetic Diversity of the Naive Human sFv Phage DisplayLibrary

33 randomly chosen sFvs were DNA sequenced to analyze genetic diversityto identify the V_(H), D, J_(H), V_(kappa), J_(kappa), V_(lambda) andJ_(lambda) germline gene segments and V_(H)CDR3 length and to create aDFCI database of recovered sFv genes. Analysis of germline gene segmentsis through “V Base: A database of human immunoglobulin variable regiongenes. Ian M. Tomlinson, Samuel C. Williams, Simon J. Corbett, JonathanP. L. Cox and Greg Winter. MRC Center for Protein Engineering, HillsRoad, Cambridge, CB2 2QH, UK”. The data from these analyses are shown inTables 1-3.

Table 1 shows the results of human V_(H) germline gene usage for 33V_(H) genes for which we could make an assignment. The diversityincludes 20 different germline genes representing five of seven V_(H)families. None of the replicate V_(H) genes (e.g. DP-875. DP-7, S12-14,etc.) are identical to other members that are derived from the sameV_(H) germline gene. Another indication of genetic diversity is thelength of the V_(H)CDR3. The data presented in Table 2 shows that theaverage length of this diversity segment ranges from 6 amino acids to 18amino acids with the majority of the rearranged V_(H) genes showing CDR3lengths between 10 and 14 amino acids. This is in excellent agreementwith published reports with natural antibodies. Finally, 28 V_(L) geneswere analyzed for V_(kappa) and V_(lambda) germline gene assignment. Inhumans, these two classes of light chains are used at a frequency ofapproximately 1:1 unlike mouse where 95% of light chains are kappafamily members. As can be seen in Table 3. eight different V_(kappa)germline genes were identified representing five of six differentV_(kappa) families and 12 different V_(kappa) germline genes were usedrepresenting eight of 10 different V_(lambda) families. Again, whenreplicate V_(L) germline gene usage occurred somatic point mutationsconfirmed that the genes were not identical.

Accordingly, we believe there is broad genetic diversity in this verylarge, naive, human sFv antibody-phage display library. Each of themajor heavy and light chain families were represented, but not all ofthe minor families. The latter finding is most likely due to the smallsample size that we have analyzed.

In Vitro Functionality of the Single-Plasmid Inducible System

A transfection assay was performed as was described in methods. Asinternal comparison for our experiment, we included a transfection assayusing the two plasmid system described by Yao et al., Human Gene Ther.9: 1939-1950 (1998). Results obtained co-transfecting pCMtetOhEGF withthe control vector, pcDNA3 or the plasmids were consistent with thefindings reported in Yao's paper (data not shown). FIG. 3 represents theresults obtained after supernatant analysis of Vero cells transfectedwith the tetracycline-inducible plasmid at various concentrations. Thereare not significant differences in term of efficiency of our system when2, 5 or 10 μg of plasmid was used for transfection. As described for thetwo plasmid system, hEGF expression was reduced in a time-dependentmanner. Notably, HEGF expression was repressed and sustained for aperiod of 72 hrs, reaching about 1,300-fold repression at 46-72 hrpost-transfection.

Three genetically modified HIV-1 based vectors (described above) weretested for their ability to infect HeLa cells in vitro. The eGFP wasused as reporter gene and gene expression driven from the internal CMVpromoter or from the viral promoter itself evaluated by FACS analysis.Table 4 shows the results obtained with the 6 constructs. There is nosignificant reduction in the titers obtained when the originallentiviral vector was self-inactivated or when a significant portion ofthe gag gene and the first exon of the tat gene were removed. It isimportant to point out that the reverse transcriptase titers obtainedwith our preparations don't differ between the different constructions(data not shown). This fact correlates to some previous reports where ithas been demonstrated that the integrity of the tat protein isfundamentally required to increase viral transactivation during viruspropagation. In our case, a full sequence of the exons 1 and 2 of thetat gene is provided in trans into the packaging construct duringtransfection, providing the necessary amount of tat protein to producethe virus. In Table 1, we can also observe that expression of eGFP canbe directed by the wildtype viral promoter (HVPΔEB). Furthermanipulations of the promoter region such the self-inactivation slightlyreduces gene expression driven by the viral promoter but, when thestrong trans-activator, tat protein, is not present, could moresignificant decrease the % of fluorescence. indicating some promoterattenuation.

Transcriptional Control of mRNA Expression by tetR

A polycistronic mRNA of about 2 Kb, encoding the reporter gene as wellas the tetR is the result of the initial rounds of gene transcriptionfrom both, inducible and control plasmids (FIG. 2). Initial productionof tetR by cap-independent translation is mediated through an IRESsequence. The ˜500 nucleotides of the IRES element contains thecis-acting elements necessary to recruit the small ribosomal subunitspromoting internal initiation of translation of RNA [E. Martinez-Salas,Curr. Opin. Biotechnol. 10: 458-64 (1999)]. Concomitantly with tetRproduction, transcriptional shut off occurs in the absence oftetracycline. The mechanism can be explained as a high affinity andeffective interaction between dimers of tetR and two tandem tetOsequences located between the TATA box and transcription start site ofthe CMV promoter, resulting in blockage of transcription initiation.When tetracycline (Tet) is added to the system, tetR releases binding tothe tetO because of a higher association constant between the repressorand the antibiotic [W. Hinrichs et al., Science 264: 418-420 (1994)]. Asa result, high levels of expression can be achieved through activationof the chimeric CMV promoter.

Transcript levels found in transduced VERO cells after 48 hrpost-transfection were analyzed by Northern blotting. A radiolabeledtetR probe was used to visualize mRNAs produced from the control andinducible plasmids (underlined in FIG. 2A). Total RNA fromnon-transfected cells and from cells transfected with an empty controlplasmid were considered our negative control (FIG. 2B, lanes 1,2). Inparallel, cells were transduced with pcDNAtetR plasmid and its RNA usedas positive control of our experiment (lane 3). The probe was able todetect a transcript of about 0.6 Kb corresponding to the mRNA size ofthe tetR gene. Cells transduced with a one-piece control plasmid (1Pc)showed expression of a higher molecular weight mRNA corresponding to theexpected size for our construct. No differences in expression can benoted in the absence or presence of 1 mg/ml tetracycline (FIG. 2B, lanes4,5). However, transcript levels corresponding to cells transduced withthe one-piece inducible cassette (1Pi) showed regulation of expressionaccording to the proposed model (FIG. 2B, lanes 6,7). In the absence ofthe antibiotic and 48 hours post-transfection, no transcript could bedetected. As we described before, a few molecules of bicistronic mRNAneed to be synthesized to serve as a mold for cap-independenttranslation of the repressor. It is possible that the initial mRNAmolecules get rapidly degraded from the cell, or that low levels of mRNAare being produced at very basal levels to maintain silenced genetranscription. Another possibility is that the life-span of tetR in thecell is long enough to preserve de-regulation of the system. Total RNAlevels are shown in the bottom panel of FIG. 2B, demonstrating thatequal amounts of RNA were loaded.

Regulation of hEGF Expression from a Single Cassette

Tight control of gene expression requires a system with highinducibility, specific and dose-dependent response to the inducer, aswell as the capability to return to basal levels after the inducer isremoved. We tested these three properties of the single cassette by invitro transfection experiments.

Efficiency. We analyzed the efficiency of our single cassette compare tothe previous system described by Yao and collaborators. [F. Yao et al.,Human Gene Ther. 9: 1939-1950 (1998)], where the expression control andthe regulatory components are present in two separate plasmid. For thatpurpose, we performed parallel functional studies of the efficiency ofboth systems by measuring the amount of hEGF secreted into the culturemedia of transfected VERO cells (FIG. 7). Experimental conditions weresimilar to those described for the two plasmid system [F. Yao et al.,Human Gene Ther. 9: 1939-1950 (1998)]. Reporter gene expression wasanalyzed after harvesting the extracellular medium every 24 hr andmeasuring the amount of HEGF produced by ELISA. The data obtained usingthe two plasmid construct were consistent with the results reportedpreviously by Yao et al., Human Gene Ther. 9: 1939-1950 (1998).Expression of hEGF from the control plasmid did not exhibit anyvariation to antibiotic administration. Expression of hEGF frompCMVtetOhEGF was unaffected unless tetR was co-transfected achievingabout 340-fold repression during the first 24 hr. increasing up to600-fold and 950-fold during the two consecutive time points,respectively, in the absence of tetracycline. Similarly, using oursingle cassette, we observed no difference in hEGF expression levelsdriven out of the CMV promoter of the 1Pc construct. However, atime-dependent tetR repression was clearly observed using the 1Pisystem. 55-fold, 100-fold and 900-fold repression were detected at 0-24hr, 24-48, and 48-72 hr post-transfection. Both genes weresimultaneously expressed from the bi-cistronic mRNA during the firstround of transcription until sufficient IRES-mediated tetR synthesis wasachieved to block gene activation. Consequently, cap-mediatedtranslation of the first cistron occurred, contributing to higher levelsof hEGF production from the 1Pi system compare with the two plasmidsystem in the absence of tetracycline. We also observed thattetR-mediated repression using the single cassette exhibited a certaindelay compared to the results observed using the two plasmid system. Theexplanation could be that the required levels of tetR are not reacheduntil 48 hr post-transfection, and/or the complete clearance of theinitially synthesized hEGF occurs over time. After 48 hr comparablelevels of repression were obtained from both systems proving theefficiency of our system.

Dose-response. Release of tetR-mediated repression was observed afteraddition of increasing concentrations of tetracycline to the culturemedia of transfected VERO cells with the 1Pi system (FIG. 8). Fullactivation of the system was obtained with 50 ng/ml of tetracycline.

Reversibility. We have tested the capability of the 1Pi system torespond to tetracycline removal after induction (FIG. 9). Vero cellstransfected with the 1Pi construct were incubated in the absence orpresence of 1 μg/ml of tetracycline. After 24 hr, a set of cellspreviously exposed to the inducer were refed with fresh medium lackingtetracycline and the concentration of hEGF was analyzed in the culturemedia. As shown in FIG. 9, hEGF secretion continued almost unaffectedover the next 24 hr but dramatically dropped to basal levels after 48 hrin the absence of tetracycline. Transcription initiation of the hEGFgene in cells previously undergoing gene expression was inhibited,achieving 2.500-fold repression for at least 2 days. Cells that werekept in the uninduced state exhibited a maximum of 10,000-foldrepression after 3 days post-transfection.

Regulation of eGFP Expression from a Single Plasmid System in DifferentCell Lines

The ability of the tetO-bearing CMV promoter to control expression ofthe reporter gene in cell lines besides the VERO cells was determined.For that purpose, we replaced the HEGF gene from our constructs with theenhanced green fluorescent protein (eGFP) gene and screened diversecells lines for endogenous expression of the protein using FACS analysisto measure expression of eGFP at different time points (FIG. 10). In allthe cases, control cells or cells transfected with an empty vector didnot show any significant fluorescence background. (FIG. 10 shows datafor VERO cells.) VERO, COS-1, and COS-7 cells exhibited similar levelsof eGFP expression from the 1Pc plasmid, as measured by fluorescenceintensity. eGFP expression in the human cell line 293T was 2 timeshigher than in the monkey cell lines. Overall, no variation in terms ofmean fluorescence in the absence or presence of the tetracycline wasobserved. Performance of the control tetO unit was similar between celllines, reaching 5-fold repression of eGFP intensity in the absence ofthe tetracycline. The activity of the tetO-bearing CMV promoter variedbetween cell lines. In particular, higher expression levels as well asbackground in the absence of tetracycline were observed in the human293-T cell line; probably due to the presence of the E1A/B gene productsfrom adenovirus, which have been shown to promote the activity of theviral CMV promoter. Similar results were collected after analyzing cellsharvested at 24 hr, 48 hr and 72 hr post-transfection (data not shown).

All the cell lines studied exhibited significant background levels ofexpression in the absence of tetracycline. To examine whether the basallevels of expression were a consequence of leakage of the system ormerely caused by slow turnover of the eGFP protein, we usedimmunohistochemistry to look simultaneously at the production of eGFP(FITC filter) and tetR (PE filter) in transduced VERO cells without orwith the addition of tetracycline (FIG. 11). Cap-mediated-eGFP (FIGS.11A, 11C) and IRES-mediated tetR (FIG. 11B, 11D) production from the 1Pcplasmid remained unaffected in the absence or presence of the inducer.Cytoplasmic and nuclear distribution of the both proteins was observedin different cells. being mostly nuclear for eGFP and mostly cytoplasmicfor the repressor. Cells transfected with the 1Pi construct exhibited adifferent behavior. Although in the absence of tetracycline eGFP proteincould be visualized (FIG. 11E), expression of tetR was faintly observed(FIG. 11F). Moreover, when the repression was released by addingtetracycline, eGFP and tetR positive cells were detected (FIGS. 11G,11H). Therefore, tetR-mediated repression works efficiently in thosecells but the long-life and stability of eGFP does not allow us todetermine precisely in a short period of time the grade of activation orrepression of the system using this marker gene.

Introduction of a Nuclear Localization Signal Accelerates tetR-MediatedRepression

Having observed that tetR distribution is mostly cytoplasmic, a nuclearlocalization signal (NLS) was introduced at the 3′ end of the tetR geneto encourage its import into the nucleus and consequently reinforce thetetR-mediated repression of transcription. FIG. 12 illustrates theresults of transient transfection experiments in VERO cells using the1Pi system and the modified version containing the NLS sequence.Measurement of the HEGF produced and secreted into the mediumdemonstrated that no significant difference was seen between theplasmids after 24 hours. However, a more rapid tetR-mediated repressionwas observed after 48 hours with the NLS construct, obtaining 300-foldrepression or 3 times higher efficiency of the tetRNLS protein than theuntargeted tetR, in the absence of the tetracycline. After 48 hours,300-fold and 500-fold repression was achieved from the 1Pi and 1PiNLSplasmids respectively. No significant difference in terms of inductionof the system was observed between constructs. All constructs containingthe wild-type CMV promoter did not show any regulatory effectsthroughout the experiment.

Distribution of tetR in different constructs was analyzed byimmunocytochemistry using the monoclonal antibody against bacterial tetRand detecting the binding using a secondary antibody labeled with FITC(FIG. 13). Cells transfected with an empty vector showed no staining(FIG. 13A). As expected, cells transfected with pcDNAtetR were positive(FIG. 13B). To detect tetR production, cells were treated withtetracycline for 2 days prior to fixation. TetR expression from 1Pi waspresent in both the cytoplasm and the nucleus (FIG. 13C), while tetRNLSprotein was mostly found into the nucleus (FIG. 13D).

Infection of CD8-Depleted Monkey and Human PBMC's Using HIV-1 or SHIVGFP Vectors

Cell were infected using VSV-G pseudotyped HIV-1 (67,000 RT/ml) or SHIV(6.000 RT/ml) GFP viruses in the presence of 20 μg/ml of DEAE-dextranfor 4 hr. Then, the cells were washed with 1×PBS and refed with freshmedia. Green fluorescent gene expression was analyzed 48 hr.post-infection by FACS analysis.

FIG. 14 shows the results of infection of PPMC's by HIV-1 and SHIVpseudotyped VSV-G. Although GFP expression from infected monkey cells isnot as bright as the expression obtained using HIV-1 vectors in humanPBMC's, a higher percentage of fluorescent cells could be obtained usingthe SHIV viruses (about 10% with SHIV vectors versus 0.25% using theHIV-1 vectors). It is important to highlight that the amount of totalvirus used for infection differs in almost 10-fold. The use ofcomparable load of virus during infection would provide a better ideaabout the performance of the SHIV vectors compared to HIV-1 vectors inin vitro experiments using monkey-derived PBMC's. Monkey CD8-Human % ofGFP CD 8- cells/H non- 48.5 26 0.02 1 infected HIV C 40 0.37 HIV GFP 1661250 0.89 21 67 SHIV C 29.3 40 1.23 1.85 SHIV GFP 169 90 9.2 12 7

ALL REFERENCES DESCRIBED HHREIN ARE INCORPORATED HEREIN BY REFERENCE

TABLE 4 INTERNAL TAT % TOTAL VECTOR PROMOTER EXPRESSION FLUORESCENCEHVPΔEB + + 17 HVPΔEB ΔLTR + + 16.3 HVPΔEB Δtat + − 13.3 HVPΔEB − + 4.4HVPΔEB ΔLTR − + 3.8 HVPΔEB Δtat − − 2.1ALL THE REFERENCES DESCRIBED HEREIN ARE INCORPORATED BY REFERENCE

1-10. (Cancelled)
 11. A vector system comprising a first vectorcontaining a lentiviral gag gene encoding a lentiviral gag protein,wherein the primate lentiviral gag gene is operably linked to a promoterand a Polvadenylation sequence; a second vector containing an env geneencoding a functional envelope protein, wherein the env gene is operablylinked to a promoter and a polvadenylation sequence; a lentiviral polgene encoding a lentiviral pol protein on the first or second vectors oron at least a third vector, wherein said lentiviral pol gene is operablylinked to a promoter and a polyadenylation sequence; a) wherein said atleast first, second and third vectors do not contain sufficientnucleotides to encode the lentiviral gag and pol and the envelopeprotein on a single vector; and b) wherein said vectors do not containnucleotides of the lentiviral genome referred to as a Packaging segmentto effectively package lentiviral RNA; and c) wherein the lentiviralproteins and the envelope protein when expressed in combination form alentivirus virion containing an envelope protein around a lentiviralcapsid; a packaging vector containing a nucleic acid sequence encoding adesired molecule, wherein the desired molecule is an angiogenic protein,where the nucleic acid sequence is operably linked to an induciblePromoter and a lentiviral packaging sequence including portions oflentiviral long terminal repeat (LTR) sequences necessary to package thelentiviral RNA into the lentiviral virion: and d) wherein said packagingvector contains a deletion of the U3 portion of the lentiviral LTRsufficient to inactivate the lentiviral Dromoter and/or do not express atat protein that has transactivating functions; wherein the envelopeprotein is encoded by an Ebola virus gene: and wherein the lentivirus isthe human immunodeficiency virus.
 12. The vector system of claim 1 1,wherein the angiogenic protein is VEGF.
 13. A method of delivering anangiogenic protein to a vascular endothelial cell which comprisesadministering a particle produced by the vector system of claim 11.14-22. (Cancelled)